Compositions and methods to produce stable soil organic matter

Abstract

This invention is directed to compositions and methods to produce stable soil organic matter.

Description

COMPOSITIONS AND METHODS TO PRODUCE STABLE SOIL ORGANIC MATTER

[0001] For countries that permit incorporation by reference, all patents, patent applications and publications cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted being prior art.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This international application claims the benefit of priority to U.S. Provisional Application No. 63 / 634,194, filed on April 15, 2024, entitled “COMPOSITIONS AND METHODS TO PRODUCE STABLE SOIL ORGANIC MATTER” and to U.S. Provisional Application No. 63 / 677,765, filed on July 31, 2024, entitled “COMPOSITIONS AND METHODS TO PRODUCE STABLE SOIL ORGANIC MATTER.”GOVERNMENT INTERESTS

[0004] This invention was made with government support under DE-AC52-07NA27344 awarded by the Department of Energy National Nuclear Security Administration, and N00014- 21-1-2362 awarded by the Office of Naval Research. The government has certain rights in the invention.INCORPORATION OF SEQUENCE LISTING

[0005] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: 2967398-000002-Wol_SL.xml, date recorded: April 15, 2025, file size 14 kb).FIELD OF THE INVENTION

[0006] This invention is directed to compositions and methods to produce stable soil organic matter.BACKGROUND OF THE INVENTION

[0007] Soil organic carbon is the largest terrestrial reservoir of carbon and can be a target for the sequestration and management of atmospheric CO2. Sequestering atmospheric carbon into soil systems improves farmland fertility, restores degraded ecosystems, and mitigates warming.SUMMARY OF THE INVENTION

[0008] Aspects of the invention are drawn towards a method of producing synthetic mineral associated organic matter (MAOM), the method comprising: synthesizing a biofilm by culturing an orgasm under biofilm-forming conditions, wherein the biofilm produces a biopolymer scaffold, and incubating the biofilm Biofilms were synthesized in minimal medium MSgg: 5 mM KPO4 buffer (pH 7, for 1 M KPO4 in 100 mL: 9.343 g K2HPO4 and 6.309 g KH2PO4), 100 mM MOPS (pH 7), 2 mM MgC12, 700 uM CaC12, 50 uM MnC12, 50 uM FeC13, 1 uM ZnC12, 2 uM thiamine, 50 ug / mL tryptophan, 50 ug / mL phenylalanine, 0.5% glutamate, 0.5% glycerol. This is a standard medium for B. subtilis biofilm synthesis. KPOwith a mineral or salt, wherein the mineral or salt interacts with the biopolymer scaffold, thereby producing synthetic MAOM. In embodiments, the organism comprises a bacteria. The bacteria can comprise B. subtilis. The mineral or salt can be an iron mineral or salt. In embodiments, the iron mineral or salt is selected from the group consisting of iron ferric sulfate (Fe2(SO4)3), ferric chloride (FeCh), ferric nitrate (Fe(NOs)3), Ferrihydrite ((Fe3+)2O3 O.5H2O), Lepidocrocite (y- FeO(OH)), Schwertmannite (Fe8O8(OH)e(SO4) • n H2O), or a combination thereof. In embodiments the biofilm-forming conditions comprise cell density, lack of agitation, a minimal bio-film promoting media, age, or a combination thereof. In embodiments, the biopolymer comprises any one or more of TasA, TapA, exopolysaccharides, y-PGA, and BslA. In embodiments, the exopolysaccharide is selected from the group consisting of glucose, N- acetyl glucosamine, poly-P-l,6-N-acetyl-D-glucosamine (PNAG), or galactose. In embodiments, the biofilm-forming conditions comprise an increased concentration of Mn2+. In embodiments, the B. subtilis is selected from the group consisting of B. subtilis DK 1042, B. subtilis 3610, or B. subtilis 168. In embodiments, the method further comprises increasing C:N ratio of biofilm by adding glycerol to the B. subtilis during culturing. In embodiments, the B. subtilis comprises genetically modified B. subtilis. In embodiments, the B. subtilis is genetically modified to partially, substantially, or completely delete, silence, inactivate, or down-regulate a gene or fragment thereof that encodes for TasA, TapA, BslA, SipW, EpsA-O,PgsA-E, CypX-YvmC, or any combination thereof. In embodiments the B. subtilis is genetically modified to express one or more iron (oxyhydr)oxide-binding peptides), thereby increasing mineral binding affinity of the biofilm. In embodiments, the one or more iron (oxyhydr)oxide-binding peptides) comprises D6, DIO, E6, E5, (TH)n, RRTVKHHVN, or LHHLLHLLHHLLHL.

[0009] Aspects of the invention are drawn towards a genetically modified microorganism, wherein the genetically modified microorganism partially, substantially, or completely deleting, silencing, inactivating, or down-regulating a gene or fragment thereof that encodes for TasA, TapA, BslA, SipW, EpsA-O, PgsA-E, CypX-YvmC, or any combination thereof, and thereby is capable of synthesizing a biofilm that produces a biopolymer scaffold, wherein the genetically modified microorganism is genetically modified to express one or more iron (oxyhydr)oxide-binding peptides), thereby increasing mineral binding affinity of the biofilm, or both. In embodiments, said microorganism is a bacterium selected from B. subtilis. In embodiments, the microorganism comprises Atas A- sip W -tap A, AbslA, AepsA-O, ApgsBCAE, AcypX-yvmC, or any combination thereof. In embodiments, the one or more iron (oxyhydr)oxide-binding peptides) comprises D6, DIO, E6, E5, (TH)n, RRTVKHHVN, or LHHLLHLLHHLLHL.

[0010] Aspects of the invention are drawn towards a biofilm produced by a method described herein or a genetically modified organism described herein.

[0011] Aspects of the invention are drawn towards synthetic MAOM produced by a method described herein or a genetically modified organism described herein.

[0012] Other objects and advantages of this invention will become readily apparent from the ensuing description.BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1 shows an exemplary schematic of processes described herein. Fixed CO2 is introduced into the soil biome as plant litter or root exudates. These carbon-rich substrates support complex microbial communities that eventually form particulate organic matter (POM) and mineral-associated organic matter (MAOM), the former by aggregation and the latter by mineral sorption and coprecipitation. To engineer synthetic MAOM, we can use two complementary strategies that result in distinct organomineral interactions.

[0014] FIG. 2 shows a non-limiting exemplary summation of methods described herein. BslA, EPS, y-PGA, and TasA are components of the Bacillus subtilis biofilm.

[0015] FIG. 3 shows non-limiting, exemplary schematics and data described herein. Panel A shows that >. subtilis 168 biofilms are scalable. Panel B shows thatsubtilis 168 biofilms are tunable by altering the amount of glycerol in the culture medium. Panel C shows that TasA is a protein scaffold for SpyTag003 that can bind to a SpyCatcher-mRFP fusion protein. The intensity of mRFP signal is correlated with distance from the cell, proving that short peptides can be secreted using TasA as a protein scaffold, scale = 10 pm.

[0016] FIG. 4 shows non-limiting, exemplary sequences described herein. BLOSUM62 alignment of miscellaneous biomineralization proteins. Color was assigned to >90% homology. The ExxE motif binds to iron.

[0017] FIG. 5 shows non-limiting, exemplary schematics and data described herein. Panel A shows a schematic of the peptide tag engineering solution. Panel B shows agarose gel indicating insertion of mineral binding tags in the genome. Panel C shows XRD indicating the quality of lab-synthesized ferrihydrite (inset). Panel D shows biofilms from gel B incubated with hematite. Panel E shows a schematic of the biofilm engineering solution. Panel F shows agarose gel verifying genomic deletions of biofilm synthesis proteins. Panel G shows biofilm phenotypes of genomic knockouts, scale = 1 cm.

[0018] FIG. 6 shows non-limiting, exemplary illustrations of soil carbon pathways. Panel A shows a role of microbial respiration in carbon decomposition. Panel B shows the sizes of terrestial and atmospheric carbon reservoirs and their annual carbon uptake and release rates.

[0019] FIG. 7 shows non-limiting, exemplary schematics of MO AM. Panel A shows an illustration of the heirachical assembly of MAOM adapted from Zhao et al. Strong mineralogic control of soil organic matter composition in response to nutrient addition across diverse grassland sites. Science of the Total Environment. Vol. 736, 2020. Panel B shows a graph indicating that MAOM does not increase proportionally with soil organic carbon (SOC)... adapted from Cotrufo, M.F., et al. Soil carbon storage informed by particulate and mineral- associated organic matter. Nat. Geosci. 12, 989-994 (2019).

[0020] FIG. 8 shows a non-limiting, exemplary photograph of microbial growth in the presence of soluble mineral material.

[0021] FIG. 9 shows a non-limiting, exemplary illustration of a comparison of POM and MAOM from Lavallee JM, Soong JL, Cotrufo MF. Conceptualizing soil organic matter into particulate and mineral-associated forms to address global change in the 21st century. Glob Change Biol. 2020; 26: 261-273.

[0022] FIG. 10 shows a non-limiting, exemplary photograph of microbial growth.

[0023] FIG. 11 shows a non-limiting, exemplary SEM image of Bacillus subtilis 168 biofilms with biomineralized iron oxides.

[0024] FIG. 12 shows non-limiting, exemplary XRD spectra of biofilm and biofilm with mineral.

[0025] FIG. 13 shows non-limiting, exemplary13C NMR spectra.

[0026] FIG. 14 shows non-limiting, exemplary schematics and photographs of altering biofilm chemistry. Panel A shows Bacillus subtilis biofilm formation and social interactions, adapted from Amaouteli S, et al., Nat Rev Microbiol. 2021 Sep;19(9):600-614. doi: 10.1038 / s41579-021-00540-9. Epub 2021 Apr 6. PMID: 33824496 Panel B shows a nonlimiting, exemplary scarred knockout scheme that can be used to generate biofilm knockouts.

[0027] FIG. 15 shows non-limiting, exemplary photographs of biofilms with Fe2(SO4)3 and without.

[0028] FIG. 16 shows non-limiting, exemplary illustration of 513C%.

[0029] FIG. 17 shows non-limiting, exemplary graphs of MAOM consumption. Panel A shows the d13C for headspace CO2 across incubation experiments with a known bacterial consortium in rich medium (LB). The biofilms are isotopically lighter than LB. The results indicate that while biofilms are readily consumed by the bacterial consortium resulting in a lighter d13C headspace CO2, mineralized biofilms remain preserved during the course of the incubation experiment, as shown by isotopic similarity to our medium only control that has no biofilm substrate in it. Panel B shows the same incubation experiment in Panel A conducted in minimal medium (GMM) with no additional carbon added. Under minimal medium / carbon stress conditions, consumption of the biofilm is observed regardless of whether the carbon is biomineralized or not, as indicated by the lighter d13C headspace CO2.

[0030] FIG. 18 provides non-limiting, exemplary photographs showing biofilm comparison between cells with (left) and cells without (right) a pulcherriminic acid biosynthesis cluster.

[0031] FIG 19 shows non-limiting, exemplary13C NMR spectra demonstrating different biofilm carbon chemistries along a glycerol gradient. Spectra are normalized to the carbonyl peak at ~ 170 ppm. 160-220 ppm depict carbonyl functional moieties and 45-110 ppm depict O-alkyl functional moieties. We observe large variations in the O-alkyl range across a glycerol gradient.DETAILED DESCRIPTION OF THE INVENTION

[0032] Abbreviations and Definitions

[0033] Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0034] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0035] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0036] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0037] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises,” “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b, and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0038] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0039] Aspects of the disclosure are drawn towards a method of producing synthetic mineral associated organic matter (MAOM), the method comprising: synthesizing a biofilm by culturing bacteria in biofilm-forming conditions, wherein the biofilm produces a biopolymerscaffold; and incubating the biofilm with a mineral or salt, wherein the mineral or salt interacts with the biopolymer scaffold, thereby producing synthetic MAOM.

[0040] A non-limiting example of this process can be to use MSgg medium at 30 C and incubate the bacteria until a certain cell density and age is achieved. For example, the cells can be kept stationary for about 7 days for biofilm synthesis and then for another about 3 days for biomineral formation.

[0041] As used herein, the term “interacts” can be used interchangeably with the terms “binds” or “associates.” As used herein, the term interacts can refer to a covalent or a non-covalent interaction.

[0042] As used herein, the term mineral associated organic matter (MAOM) can refer to high density organic matter associated with minerals. For example, the term “synthetic MAOM” can refer to organomineral composites. In embodiments, the synthetic MAOM can be microbial MAOM or plant-derived MAOM. As used herein, the term “synthetic” can refer to a composition made by human intervention. As used herein, the term “biopolymer” can refer to any polymer that can be produced or derived from a biological system. The biopolymer can be produced or derived from a biological system, synthetically produced or derived, or a combination thereof.

[0043] As used herein, the phrase “agronomic soil amendment” can mean any material configured to be added to soil to improve the soils physical or chemical properties for the purpose of improving plant growth, increasing crop yield, improving the health of existing plant life in the soil, improving soil fertility, improving aeration, improving the water retentive properties of the soil, or any combination thereof.

[0044] As used herein, the word “organism” can refer to any organism that is capable of living in soil. In certain embodiments, the organism is an organism that is naturally found in soil. In embodiments, “organism” refers to a genetically engineered variant of a soil-dwelling microbe. The word “organism” can refer to a microbe. In one embodiment “organism” refers to a microbe that is capable of forming a biofilm. In embodiments “organism” refers to any microbe that can grow on waste biomass, e.g., lignocellulose, or directly from CO2 (autotrophs). In embodiments, the microbe is bacteria. The bacteria can comprise any bacterium is genetically tractable, capable of synthesizing a biofilm. The bacteria can be selected from the group consisting of Bacillus sublilis. Pseudomonas putida, Pseudomonas fluorescens. Komagataeibacter hansenii. Paenibacillus polymyxa, or any combination thereof. In certain embodiments, the organism comprises any bacteria within any one or more of the followinggenera: Pseudomonas, Azotobacter, Xanthomonas, Gluconacetobacter, Bacillus, and Paenibacillus .

[0045] In embodiments, the mineral or salt is an iron mineral or salt. The iron mineral or salt can be an iron oxide or an iron oxyhydroxide. The iron mineral or salt can be a poorly crystalline iron oxyhydroxide In embodiments, the iron mineral or salt can be selected from the group consisting of iron ferric sulfate (Fe2(SO4)3), ferric chloride (FeCh), ferric nitrate (Fe(NO3)3), ferrihydrite ((Fe3+)2O3 O.5H2O), lepidocrocite (y-FeO(OH)), schwertmannite (Fe8O8(OH)e(SO4) • n H2O), or any combination thereof. In certain embodiments, the mineral or salt comprises a non-iron mineral or salt. In embodiments, the mineral or salt comprises a calcium salt, a magnesium salt, an aluminum salt, or a combination thereof.

[0046] As used herein, the phrase “biofilm-forming conditions” can refer to any conditions suitable for producing a biofilm. Any biofilm-forming conditions known in the art can be used. For example, film forming conditions can be induced under a certain cell densities, stress, and age. For example, bacterial cultures can be kept stationary to produce biofilms. For example, the medium MSgg can be used to induce biofilm-forming conditions (see, e.g., Bucher T, Kartvelishvily E, Kolodkin-Gal I. Methodologies for Studying B. subiilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. .1 Vis Exp. 2016 Oct 9;(116):54612. doi: 10.3791 / 54612. PMID: 27768058; PMCID: PMC5092178.)

[0047] In embodiments, the biofilm-forming conditions comprise cell density, lack of agitation, a minimal bio-film promoting media, biofilm age, or a combination thereof. For example, the biofilm promoting conditions can comprise growth media that is cell-dense (closely packed cells). In embodiments, cell-dense conditions improve the formation of biofilm. In embodiments, to achieve cell-dense biofilm-forming conditions, the growth medium is incubated with bacteria until a certain cell density is achieved. For example, the cell density can comprise about 6 X 10e8. For example, the cell density can comprise less than about 6 X 10e8. The cell density can be more than about 6 X 10e8.

[0048] For example, the age of the biofilm can comprise about 2 to about 3 days grown at about 30°C. For example, the age of the biofilm can comprise about 0.25 days, about 0.5 days, about 0.75 days, about 1 day, about 1.05 days, about 1.1 days, about 1.20 days, about 1.25 days, about 1.3 days, about 1.5 days, about 1.75 days, about 2 days, about 2.05 days, about 2.15 days, about 2.2 days, about 2.25 days, about 2.3 days, about 2.4 days, about 2.5 days, about 2.6 days, about 2.7 days, about 2.8 days, about 2.9 days, about 3 days, about 3.05 days, about 3.1 days, about 3.2 days, about 3.3 days, about 3.4 days, about 3.5 days, about 3.6 days,about 3.7 days, about 3.8 days, about 3.9 days, about 4 days, about 4.5 days, about 5 days, or greater than about 5 days.

[0049] In embodiments, the biopolymer is can comprise any one or more of TasA, TapA, exopolysaccharides, y-PGA, and BslA.

[0050] In embodiments, the exopolysaccharide is selected from the group consisting of glucose, N-acetylglucosamine, poly-P-l,6-N-acetyl-D-glucosamine (PNAG), or galactose.

[0051] In embodiments, the biofilm-forming conditions comprise an increased concentration of Mn2+. In certain embodiments, the concentration of Mn2+is increased as compared to traditional growth media. The concentration of Mn2+can be increased prior to incubation, during incubation, or a combination thereof. The concentration of Mn2+can be increased as compared to lysogeny broth (LB) or similar grown media suitable for supporting bacteria. In certain embodiments, the concentration of Mn2+is increased by adding about 50 uM Mn2+ (as MnC12) to the to the growth media prior to or during incubation.

[0052] In embodiments, the method can further comprise increasing C:N ratio of biofilm by adding glycerol the bacteria culture, the growth media, or both. In embodiments, a 10-fold increase in glycerol permits the synthesis of biofilms with 3-fold C:N ratio variation (from ~10 to ~30). In certain embodiments an increase from 0.5% to 5% of MSgg causes a 3-fold increase in the C:N ratio. In certain embodiments combustion elemental analysis permits quantification of the C and N in the samples.

[0053] In embodiments, the B. subtilis is selected from the group consisting of B. subtilis DK 1042, B. subtilis 3610, or B. subtilis 168.

[0054] In embodiments, the B. subtilis comprises genetically modified B. subtilis.

[0055] In embodiments, the B. subtilis is genetically modified to partially, substantially, or completely delete, silence, inactivate, or down-regulate a gene or fragment thereof that encodes for TasA, TapA, BslA, SipW, EpsA-O, PgsA-E, CypX-YvmC, or any combination thereof. For example, the B. subtilis can be genetically modified to express one or more iron (oxyhydr)oxide-binding peptides), thereby increasing mineral binding affinity of the biofilm.

[0056] Aspects of the disclosure are drawn towards a genetically modified microorganism, wherein the genetically modified microorganism partially, substantially, or completely deleting, silencing, inactivating, or down-regulating a gene or fragment thereof that encodes for TasA, TapA, BslA, SipW, EpsA-O, PgsA-E, CypX-YvmC, or any combination thereof, and thereby is capable of synthesizing a biofilm that produces a biopolymer scaffold, wherein the genetically modified microorganism is genetically modified to express one or more iron(oxyhydr)oxide-binding peptides), thereby increasing mineral binding affinity of the biofilm, or both.

[0057] In embodiments, said microorganism is a bacterium selected from B. subtilis. For example, the microorganism comprises AtasA-sipW-tapA, AbslA, AepsA-O, ApgsBCAE, AcypX-yvmC, or any combination thereof.

[0058] In embodiments, the one or more iron (oxyhydr)oxide-binding peptides) comprises D6, D10, E6, E5, (TH)n, RRTVKHHVN, or LHHLLHLLHHLLHL.

[0059] Aspects of the invention are drawn towards a biofilm produced a method described herein or by a genetically modified organism described herein.

[0060] Aspects of the disclosure are drawn towards MAOM produced by the methods described herein or by a genetically modified organism described herein.

[0061] Certain aspects of the disclosure relate to use of the systems and methods disclosed herein for improved soil carbon sequestration, improved soil water properties, improved soil nutrient retention, or any combination thereof.

[0062] Aspects of the invention are drawn to an agronomic soil amendment material.

[0063] Other Embodiments

[0064] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0065] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.EXAMPLES

[0066] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1

[0067] Customizable microbial production of stable soil organic matter

[0068] Disclosed herein are systems and methods for rapidly creating a form of environmentally stable soil carbon. In embodiments, the present disclosure uses laboratorymicrobes to express a naturally occurring form of soil carbon termed “mineral-associated organic matter” (MOAM). In nature, this form of carbon is a crucial component of soils, and when present, delivers a number of ecosystem services, including soil carbon sequestration, improved soil water properties, and improved soil nutrient retention. In nature MOAM occurs in small amounts and is produced very slowly by soil microbes. In embodiments, the present disclosure relates to a microbial tool that rapidly produces this material such that it can be amended to soils. Without wishing to be bound by theory, it can be sold as a carbon offset and as an agronomic soil amendment.

[0069] Without being bound by theory, this technology creates stable C by inducing biomineralization with Bacillus subtilis biofilms. In one embodiment, Bacillus subtilis sp 168 is engineered using double-crossover homologous recombination to produce biofilms of different biopolymer compositions. These biofilms can be incubated with ferric sulfate salts for about 5 days to induce biomineralization of iron oxides. In embodiments, after iron oxides are formed on the surface, the biofilms turn a dark brown, or a combination thereof, the biofilms are harvested, washed three times in deionized, distilled water, and lyophilized for about 8-12 h or until completely dry. These biofilms can be examined for quality control using Scanning Electron Microscopy, quantified for carbon content using Combustion Elemental Analysis, and analyzed for carbon functional moieties using Nuclear Magnetic Resonance. These biofilms can be tested for resistance to degradation by other organisms.

[0070] Without being bound by theory, the choice of organism and the metal salts used to create the biofilm can be modified. For example, and without being bound by theory, Bacillus subtilis NCIB 3610, a native soil isolate, can be used to generate biofilms variants that can be treated similarly to Bacillus subtilis 168 biofilms. Alternative microbes suitable for use in the present disclosure include those that can grow on waste biomass, e.g., lignocellulose, or directly from CO2 (autotrophs). Without being bound by theory, this step streamlines the carbon removal process, taking CO2 directly to stable soil carbon. In certain embodiments, alternate or additional salts, such as ferric nitrate and ferric chloride, can be used to promote biomineralization of iron oxides. Without being bound by theory, aluminum salts can be used to promote aluminum oxide biomineralization, which represents another pool of organomineral interactions in the soil.

[0071] In various embodiments, the presently disclosed material acts to stabilize soil carbon, creating a long-term carbon sink. The present disclosure can be used as a carbon credit by to reduce the carbon footprint of an entity (such as a corporation) or an individual. In embodiments and without being bound by theory, the present disclosure improves soil healthby stabilizing soil water and nitrogen, improving overall crop agronomic performance and ecosystem resilience.

[0072] Current microbial soil additives do not provide direct carbon offsets. In embodiments, the present disclosure permits the production of mineral-associated organic matter in a laboratory setting, which eliminates a natural bottleneck to stable soil carbon production: waiting for microbially-produced carbon to associate naturally with minerals. In the environment this process can take decades. In embodiments, the present disclosure permits the production of MAOM in the lab on the timescale of a few days.

[0073] Bench-scale experiments show organisms scale up production relatively easily.

[0074] Without wishing to be bound by theory, we can optimize our system for higher yield. We can also optimize our measurement protocols and testing conditions to determine the persistence of biofilm carbon in organomineral associates.

[0075] We will compare the chemical fingerprint of our lab MAOM and control biofilms before and after incubation in soils using13C nuclear magnetic resonance spectroscopy.

[0076] Specific compositions of organic matter (C:N ratio, molecular weight) and inorganic matter (Al3+vs Fe3+) are positively correlated with persistence of MAOM in soil, but it is unknown whether Al3+or Fe3+creates more persistent MAOM. As used herein, “persistence” can refer to the time period during which MAOM remains in the soil without significant degradation. Without wishing to be bound by theory, the present disclosure permits a) manipulation of the B. subtilis biofilm composition using genome engineering to increase the proportion of extracellular polysaccharides or proteins, which will allow for testing the relative importance of C:N ratio and molecular weight on MAOM persistence, and b) growth of the B. subtilis biofilm in the presence of Fe3+ ions or A13+ ions to study the influence of inorganic matter composition on stabilization.

[0077] Public pressure and regulation on CO2 are driving companies to find ways to offset carbon emissions. One way for companies to create carbon offsets is to store carbon in nature, typically by reforestation, returning agricultural waste to soils, or creating biochar. However, reforestation and return of agricultural waste hit a natural upper limit to the amount of carbon that can be stored, set by limited land available for forests and the very slow rate of carbon binding to soil minerals. Biochar, an emerging technology, cannot scale beyond ~1 GT of CO2 because it competes with forests and food production. In various embodiments, the presently disclosed technology removes both the storage and scalability limits of existing technologies. Moreover, the presently disclosed technology can simultaneously increase food production and support ecosystem resilience by increasing soil health. In embodiments, the presently disclosedtechnology stabilizes carbon in soils by driving association of soil minerals and biomass into stable soil carbon, is the presently disclosed technology is much faster than the natural process for producing mineral-associated organic matter, which can take multiple decades.

[0078] This technology moves atmospheric carbon into soil in a way that simultaneously removes CO2, stabilizes soil water, and increases crop productivity. Without being bound by theory, the present technology can be developed as a product for soil amendment.

[0079] The present disclosure includes products to improve crop performance, a means for creating carbon offsets to meet, or a combination thereof.

[0080] In various embodiments, the present disclosure relates to a synthetic biology tool that is not at risk of concerns about GMO release. For example, in certain embodiments, the present disclosure relates to material that is the product of a microbe, where the product is used, but not the microbe (e.g., like microbially produced insulin.) In embodiments, no DNA is added to the organism in the process. In such embodiments, for example, no recombinant DNA is added to the genome.EXAMPLE 2

[0081] Non-Limiting, Exemplary General Workflow and Method Compilation

[0082] Bacterial biofilms can be synthesized in minimal medium with glycerol as the carbon source. After 3 days, a concentrated solution of ferric sulfate (Fe2(SO4)3) was added in the test samples, with 3 mL double distilled water (ddEEO) added in the control to compensate for medium dilution. Samples were covered in foil and incubated at 30 C for 7 days to facilitate biomineralization of iron oxides.

[0083] After 7 days, samples were harvested in sterile 15 mL tubes and washed 3x with ddH2O to remove residual medium. An aliquot of the sample was harvested onto stainless steel coupons for correlative SEM / Raman. The leftover sample was lyophilized for 12 h or until dry.

[0084] Carbon-nitrogen content and13C composition of samples were determined by combustion elemental analysis. Samples were carbon-normalized and incubated in LB broth with a premixed soil consortium for degradation experiments. d13C of headspace CO2 was measured by a gas bench IRMS.

[0085] Engineering intervention - strains were engineered using double-homologous recombination in Bacillus subtilis 168 and 3610 (DK1042). The biofilm knockouts thus generated are unique and have not previously been investigated for biomineralization. The biomineralized bacterial biopolymers disclosed herein represent an improved approach tosynthesizing organomineral associations in the laboratory that can be scaled up for carbon sequestration.

[0086] Scientific intervention - understanding the mechanisms behind carbon preservation in soils has been a persisting challenge due to the chemical complexity and physical heterogeneity of centennially-preserved organomineral associates. Our model system directly probes the role of minerals in carbon protection by eliminating confounding factors such as bulk carbon chemistry, soil hydration, and soil texture.

[0087] In embodiments, the general workflow can be subdivided into three phases - synthesis, characterization, and testing.

[0088] Synthesis

[0089] Bacterial strains and plasmids

[0090] Strains were constructed in Bacillus subtilis DK1042 and 168. B. subtilis DK1042 is a mutant of the isolate B. subtilis 3610 with a coml knockout to facilitate transformation (Plasmid-Encoded Coml Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis - PMC). B. subtilis DK1042 was purchased from the Bacillus Genetic Stock Center.

[0091] An 800 bp fragment was amplified on both sides of the operons of interest to facilitate sequential knockouts by double-homologous recombination. PhantaFlash™ polymerase was used to amplify all PCR products according to manufacturer recommendations (2 x Phanta Flash Master Mix P510). Plasmids and linear products were constructed by Gibson assembly using the NEBuilder HiFi assembly system (NEBuilder® HiFi DNA Assembly Master Mix). Plasmid list (Table 4), include Tms for cPCR. See the Sequence Listing, which is filed herewith for the applicable sequences.

[0092] Table 4. Shows a plasmid list.

[0093] Bacterial and biofilm growth conditions

[0094] Cells were streaked onto LB plates and incubated at 30 C for 12 h. Single colonies were inoculated into 3 mL of LB and cultured at 30 C, 250 rpm for 8 h. Cultures were washed thrice with ddELO at 10000 g, 2 min and resuspended in 2 mL ddH2O.

[0095] Biofilms were synthesized on 10 cm petri dishes in 15 mL MSgg medium (recipe citation from Akos Kovacs, glycerol 513C) for 3 days at 30 C. 3 mL of Fe2(SO4)3 in ddH2O (3.125 mM) was added to synthesized biofilms and plates were covered in foil to prevent photochemical alterations to Fe. After 1 week, samples were harvested and lyophilized for 12 h. Approx, a fourth of each sample was reserved for SEM.

[0096] Non-Limiting Example of Minimal medium for carbon stress - GMM recipe: Genetic engineering of bacterial strains

[0097] Table 3: GMM recipe

[0098] Adjust pH to 7.0.

[0099] After autoclaving the above solution, add:

[0100] 1. MgC12.6H2O - 1 mL of 20% solution, added after autoclaving

[0101] 2. SL7 - 0.1 mL solution, added after autoclaving

[0102] Characterization

[0103] SEM

[0104] Harvested samples were preserved in 1 mL ice cold Kamovsky’s fixative and stored in 4 C for 24 h. The fixative was washed out thrice with 0.1 M HEPES (pH 7.2) for 10 min each. Washed samples were dehydrated in a graded alcohol series, with two washes at each step of the concentration gradient (25%, 50%, 70%, 95%, 100% EtOH). Samples were further incubated in 100% EtOH for 1 h and dried using a Critical Point Dryer to maintain bacterial cell structures.

[0105] Dehydrated and dried samples were mounted onto a 12.7 mm aluminium stub and coated with 10 nm carbon.

[0106] Samples were imaged in a ThermoFisher Apreo electron microscope at 5 kV with the T1 detector.

[0107] Powder XRD

[0108] Lyophilized samples were flattened onto a glass sample holder such that the sample powder completely covered the holder. X-ray diffraction spectra were determined onthe Rigaku D / Max Ultima II Powder XRD with CuKa radiation ( = 1.5406 A). Measurements were recorded at 2theta / theta scanning mode with angles ranging from 10 - 80 degrees (step?) with the following slit configuration: DivH.L.Slit = 10 mm, DivSlit = 1 deg, SctSlit = 1 deg, RecSlit = 0.30 mm.

[0109] Standard baseline subtraction was performed onto raw spectra, and processed spectral data was plotted on Origin 2021b.

[0110] Elemental analysis

[0111] Elemental combustion analysis was performed on the Costech 4010 CHNS / O Elemental Analysis System to determine C and N composition. Samples were weighed in Sn capsules (5 x 9 mm, Costech) with weights averaging ~ 0.3 mg. Acetanilide was used as a standard (0.05 mg - 3.0 mg), with phenylalanine (~ 1.0 mg) as a quality control internal standard. All samples were run in three biological replicates. Data analysis was conducted within the EA software with a linear fit calibration for Nitrogen and a quadratic fit for Carbon.

[0112] 13C Solid-state NMR

[0113] Bulk chemical composition of the samples was determined using solid state13CCP / MAS NMR. Samples were analyzed on a 200 MHz Bruker NMR spectrometer (50 MHz13C resonance frequency) equipped with a MAS probe (7 kHz spinning speed) (4 mm diameter zirconia cylindrical rotor with a Kel-F cap). Chemical shifts were calibrated to a glycine spin counting standard.

[0114] To determine the alkyl, O-alkyl, carboxylic, and aromatic fractions, collected NMR spectra were processed through a molecular mixing model (Baldock, 2004, Nelson, Baldock, 2005).

[0115] Testing

[0116] Incubation experiments

[0117] Cultures of Pseudomonas putida, Burkholderia unamae. and Rhodococcus ruber (from Ross Thy er) were mixed in 1 mL LB. A preweighed, carbon-normalized amount of synthesized organomineral associates was incubated with cultures in 30 C for 1 week. Headspace CO2 gas was measured using gas bench-IRMS.

[0118] Culture tubes - 12ml Borosilicate Glass Vial Round bottom, with white septum cap, 938W- 101x15.5mm (E2860-100).

[0119] Note: Measurements - gas IRMS determines isotopic signature of C (513C). The sample is much lighter, as it is grown on petroleum-derived glycerol (- 24 %o on average). LB typically measures at 513C = - 19 %o. This difference in C isotopic composition can be used to parse out where headspace CO2 is being released from.

[0120] Introduction

[0121] Preserved soil organic carbon can be a reservoir for carbon sequestration. Soil organic carbon is the largest terrestrial reservoir of carbon, holding more than twice the amount of carbon in the atmosphere. While a portion of this carbon is remineralized to CO2, a fraction is preserved for centennial to millennial timescales in high-density organo-mineral associations. Determining the mechanisms of carbon preservation in these organo-mineral associations is essential to soil management practices and policy interventions. However, these associations are challenging to investigate due to their chemical complexity and resistance to isolation.

[0122] Herein we describe the design of a model system for these organo-mineral associations that are synthesized in a chemically controlled, lab-scale environment. In certain embodiments, we engineered Bacillus subtilis NCIB 3610 comlQ12L, a native soil organism that produces robust biofilms, to synthesize a biopolymer scaffold upon which amorphous iron oxides are biomineralized. We characterized the mineralogy of the iron oxides thus formed and the bulk chemical composition of the enriched Bacillus subtilis biopolymers. We subsequently conducted synthetic microcosm experiments with our model organomineral associates to demonstrate the role of carbon stress in organic matter degradation. Additionally, the presently disclosed system allows for genetically encoded modifications that can customize biopolymer scaffold chemistry. By synthesizing exopolysaccharide-deficient organomineral associates, we elucidate the role of polysaccharides in long-term carbon preservation.EXAMPLE 3

[0123] Customizable synthetic proxies for stable soil organic carbon

[0124] Soil organic carbon (SOC) is the largest pool of terrestrial carbon and an essential component for the long-term management and sequestration of atmospheric CO2. A fraction of this SOC is protected by association with soil minerals, thereby rendering this carbon sequestered for millennia. Understanding this preserved pool of SOC is fundamental to soil management practices and policy interventions. However, due to its chemical complexity and resistance to isolation, stable soil carbon has been challenging to study.

[0125] Herein, we report an approach for synthesizing stable soil carbon in the laboratory. We have designed bottom-up bacterial biopolymers that act as proxies for stable soil organic carbon. These biopolymers form complexes with short-range order iron oxides on laboratory timescales and can be produced with varying protein, carbohydrate, and C / N ratios. The tunable chemistry of these organomineral composites allows for robust, lab-scale incubation studies exploring the effects of organomineral associations on soil carbon and nitrogen cycling. Thiswork can dissect the mechanisms of carbon protection, determine microbial roles in soil carbon preservation, and inform models that predict soil feedback to climate change.EXAMPLE 4

[0126] Engineering organomineral interactions to synthesize mineral-associated organic matter for long-term carbon sequestration in soils.

[0127] Abstract

[0128] Soil organic carbon is the largest terrestrial reservoir of carbon and can be a target for the sequestration and management of atmospheric CO2. Sequestering atmospheric carbon into soil systems improves farmland fertility, restores degraded ecosystems, and mitigates warming.

[0129] While some of the soil carbon is mineralized to CO2, a fraction is preserved in organomineral associations termed as Mineral Associated Organic Matter (MAOM). Increasing the amount of MAOM in soils can sequester carbon for centennial to millennial timescales. However, there is a limit on how much MAOM can be accumulated in soils due to limitations in carbon transport and the saturation of mineral surfaces. Studies on the preservation mechanisms of MAOM are sparse, as MAOM is challenging to isolate and characterize due to its chemical heterogeneity and micron size, thereby limiting our understanding of stable carbon pools in soils.

[0130] Emerging empirical evidence on the composition of MAOM highlights the role of microbial biomass and necromass in MAOM formation. The preferential enrichment of microbial biomass offers the possibility of engineering MAOM using synthetic biology. Without wishing to be bound by theory, MAOM can be engineered using the naturally synthesized biopolymers from the Bacillus subtilis biofilm as a template for organomineral interactions. These biomaterial proxies of MAOM can be used as model systems to better understand soil C protection mechanisms, elucidate microbial roles in soil C preservation, and inform models that predict soil feedbacks to climate change. In addition, synthetic MAOM is the first step towards a facile, affordable, and scalable soil amendment for carbon sequestration.

[0131] Introduction

[0132] Soil organic carbon (SOC) is the largest terrestrial reservoir of carbon, storing more than 2500 petagrams of carbon in the top 2 m alone k SOC is beneficial for agricultural soils, enhancing soil structure, increasing water-holding capacity, and inhibiting erosion2. This pool of carbon actively cycles between the atmosphere and the terrestrial biosphere, rendering it central to understanding the impact of increasing anthropogenic CO2 on soil carbon feedbacks.SOC has garnered interest as a reservoir for excess atmospheric CO2 with additional benefits that range from replenished soils and water systems to increased agricultural security3 5.

[0133] However, not all soil carbon is created equal. SOC is a chemically heterogeneous mixture of plant litter, microbial biomass, and miscellaneous organic exudates2. A portion of SOC is degraded rapidly into CO2 and released into the atmosphere. This fast-cycling fraction of SOC, or Particulate Organic Carbon (POC), is typically larger and more accessible to microbial degradation6,7The remaining SOC is protected in complex, slow cycling, organomineral associates called Mineral Associated Organic Matter (MAOM)6>8’9. MAOM is less accessible to microbial degradation due to its inherent chemistry, physical occlusion, and protection by minerals6’10"14. As a result, MAOM has a residence time of centennia to millennia in soil systems, making it of particular importance for carbon sequestration6’13,15.

[0134] MAOM has garnered significant interest for applications in the sequestration of excess atmospheric CO2. However, there are challenges to increasing the fraction of MAOM in soils- limited understanding of the chemistry of MAOM, and the inherent carbon saturation in soils16 l 8. MAOM is challenging to study due to its small size, spatial heterogeneity, and complex chemistry6. Studies of MAOM have been scarce as it is difficult to isolate and characterize MAOM from native soils. In addition, soils have a hard upper limit on mineral surfaces available for MAOM sorption and coprecipitation, resulting in a saturation of mineral surfaces and a cap on the amount of MAOM a soil can capture10,18. This gap in our understanding of MAOM and carbon preservation has led to models that are unable to precisely assess carbon turnover in our soils and, ultimately, are unable to accurately predict soil feedbacks to anthropogenic climate change15,19. Model MAOM materials can not only answer questions about emerging protection mechanisms of organic matter in soils but can also provide a scalable soil carbon sequestration strategy.

[0135] Chemically recalcitrant carbon can have longer-than-average residence times in soils. For example, the Salk Ideal Plants shuttle large amounts of carbon to their roots and form reservoirs of waxy biopolymers called suberin, or the burial of lignocellulose and bacterial nanocellulose, all of which require specialized enzymes to degrade20 23. Recalcitrant forms of plant litter, such as lignin, have also been suggested to persist for long periods in soils24While deeper root-mediated burial of recalcitrant carbon will preserve it for longer, recent studies show that such recalcitrant carbon does degrade, albeit slowly, releasing CO225 28. As a result, chemical recalcitrance is slowly being dismissed as the primary mechanism of carbon protection.

[0136] An emerging mechanism for carbon preservation in soils results from the interactions between microbial biomass and mineral surfaces4>9>15>29>30Microbes decompose carbon-rich plant litter and deposit nitrogen-rich biomass onto charged mineral surfaces, thus mediating organomineral interactions in soils by adsorption29,31. A second proposed mechanism of MAOM formation is by coprecipitation - dissolved or weathered minerals nucleate onto microbial biomass and form MAOM31,32. Iron and aluminum oxides and oxyhydroxides play a key role in OM preservation by providing a surface for sorption and OM protection10>13>33 35Short-range order, poorly crystalline iron oxyhydroxides, such as ferrihydrite, have been found to co-occur with OM in soils and readily adsorb SOM in benchtop bioreactors32,36 38.

[0137] Along with mechanisms that elucidate the importance of organomineral interactions for SOC preservation, recent work affirms the central role of microbes in SOC synthesis and preservation39 42. The carbon-to-nitrogen ratio of organic matter in soils decreases with depth, indicative of decomposition of plant litter before preservation43,44Microbial stoichiometry acts as a control on the carbon-to-nitrogen ratio of MAOM by preferentially degrading substrates with favorable C:N for biosynthesis45. In addition, the metric of microbial carbon use efficiency (CUE), the amount of carbon used for biosynthesis relative to total carbon consumed, is positively correlated with the amount of SOC in soils39. Incubations conducted with isotopically labeled microbial necromass substantiate the preferential accumulation of necromass in SOC41. This microbial necromass can cycle within the soil microbiome for multiple generations before being stabilized in organomineral complexes4I-42-46 48.

[0138] Without wishing to be bound by theory, engineered microbes can synthesize MAOM in a scalable and standardized manner. Studies on SOC preservation mechanisms use microbial necromass as a proxy for natural organic matter instead of pure organic substrates such as chitin and xanthan gum49 51. While the use of bacterial and fungal necromass satisfies the immediate requirement for SOC proxies, it does not accurately represent the central role of minerals in SOC storage. Engineered MAOM can be useful. First, there is a need for a new, standardized, model material that can serve as a template for experiments designed to understand fundamental aspects of SOC formation and degradation. Second, the production of synthetic MAOM is the first step in overcoming a fundamental limitation in soil carbon sequestration: the current hard cap on the size of the global soil MAOM carbon reservoir set by the preexisting soil mineral inventory. Without wishing to be bound by theory, engineered MAOM is can be a scalable carbon reservoir that can be used as a soil amendment. As this is a new approach to use bacterial biomass and biopolymers as a MAOM reservoir, we described hereina non-limiting set of design principles for synthetic MAOM. Employing these design principles, we can assemble a scalable, tunable, and engineerable MAOM proxy.

[0139] MAOM is sparsely studied and poorly understood. A model MAOM can act as a testbed and highlight emerging carbon protection mechanisms.

[0140] Non-Limiting, Exemplary Research Plan

[0141] We can use synthetic biology to engineer a de novo, microbially-synthesized proxy for MAOM, or Synthetic MAOM, as a testbed for soil science and prototype for future development of biomaterials for carbon sequestration. This engineered biomaterial can be designed by combining existing models of MAOM formation, native carbon-to-nitrogen ratios from soils, and empirical data on size, structure, and chemistry at the organomineral interface from natural MAOM. Synthetic MAOM will provide a framework for dissecting the various components of MAOM formation and preservation in a facile, affordable, and scalable process. De novo synthesis of MAOM can be useful to probe the effects of soil chemistry and mineral association on MAOM preservation and inform models on soil feedbacks to climate change.

[0142] Additionally, synthetic MAOM can act as a scalable biomaterial reservoir for carbon sequestration with potential applications as a safe soil amendment to improve soil structure, increase nutrient concentrations, and enhance water retention2,5.

[0143] We can design a new biomaterial proxy for MAOM in soils using synthetic biology. We can identify the ideal chassis organism and biopolymer matrix for synthetic MAOM. Subsequently, we can engineer organomineral interactions between this biopolymer as the template and poorly ordered minerals. Lastly, we can test the persistence of this new model material in synthetic and natural soil microbial communities by measuring natural isotopic ratios (513C), Stable Isotope Probing, and GC-MS or IRMS. Figure 2 illustrates a non-limiting, exemplary research plan.

[0144] Without wishing to be bound by theory we can design a bacterial platform to synthesize robust, tunable, and chemically characterized biomaterials using a GRAS chassis.

[0145] Soils contain the highest bacterial diversity of any environment, hosting 10e2 - 10e6 unique species per gram52 55. These bacteria play pivotal roles in the degradation of organic matter, synthesis biomass and biofilms, and the accumulation of microbially-derived exudates and necromass 56. Microbial compounds account for 50-80% of SOM constituents24-39-57 59. Bacterial biofilms and extracellular polymeric substances have been found in close association with minerals in soils49>50>60~65Bacterial polymers serve roles in MAOM formation - they are preferentially sorbed onto mineral surfaces, and they act as a template for mineral nucleation and biomineralization49’56’60. in effect, bacterial extracellular polymers are an organic trap forminerals in soils, making these biopolymers the ideal matrix for synthetic MAOM. I aim to exploit this natural affinity of biopolymers by using Bacillus subtilis 168, a native soil organism and robust biopolymer producer, as my chassis.

[0146] Without wishing to be bound by theory, the biofilm of Bacillus subtilis 168 is a scalable, tunable, and engineerable organic matrix for MAOM biosynthesis.

[0147] The B. subtilis 168 biofilm is scalable and tunable: B. subtilis 168 was cultured in biofilm-forming conditions, i.e., with an increased concentration of Mn2+in different volumes of rich (Luria-Bertani (LB) with glycerol) and minimal (MSgg) media. Dry biofilm weight in rich and minimal media was measured by lyophilizing synthesized biofilms after 96 h at 30 °C. In figure 3 shows that increasing the volume of both LB with glycerol and MSgg increases the production of biofilms. The increase in biofilm production is lower in MSgg, as expected.

[0148] Carbon concentrations were altered in the minimal medium and measured the C:N ratio of the resultant biomass by quantitative elemental analysis. This indicates tunability. Preferential retention of organic matter in soils is directly correlated with the C:N ratio of the resultant SOM17,44Figure 3 shows increasing the concentration of glycerol in minimal medium results in biofilms with increased C:N ratios.

[0149] The B. subtilis 168 biofilm is engineerable: By engineering the secreted biofilm protein TasA, the B. subtilis biofilm can be patterned and programmed66. TasA polymers are amyloid-like and are found in abundance in the biofilm matrix67,68. We can use TasA as a protein scaffold for various mineral-binding and biomineralization peptides. These peptides can be distributed across the biofilm by TasA and act as nuclei for mineral adherence. We secreted a genomically integrated tasA-SpyTag system and incubating the biofilm with purified SpyCatcher-mRFP protein69. The resulting biofilm was visualized using optical microscopy. We can use SpyTag as a model for short mineral peptides.

[0150] In embodiments, engineering BslA, a surface-level hydrophobic biofilm protein can be used to avoid occlusion by other matrix polymers. In some embodiments, B. subtilis NCIB3610 comIQ12L can be used to increase its competence. Genetic competence, or simply “competence” can refer to the ability of cells to bind to and to take up exogenous DNA. NCIB3610 comIQ12L increases competence in NCIB3610. NCIB3610 is an attractive chassis option as it is a direct soil isolate and has not been engineered for lab use. NCIB3610 comIQ12L is a mutation in NCIB3610 that makes it more competent.

[0151] Without wishing to be bound by theory, we can engineer organomineral interactions at the biofilm-mineral interface using mineral- binding peptides and biofilm engineering.

[0152] Knowledge gaps in natural MAOM formation make it challenging to determine which mechanism of microbe-mineral interactions is essential for MAOM synthesis. To address this gap, we can adopt complementary approaches to facilitate organomineral interactions - through mineral-binding tags and rational biofilm engineering. Incubating bacterial biofilms with lab- synthesized poorly ordered minerals is one approach - the resulting material can be an experimental control for sorption-mediated MAOM formation. Incubating bacterial biofilms and necromass with saturated solutions of soluble Fe3+salts is another approach - the resulting material can be an experimental control for coprecipitation-mediated MAOM formation. The ability to synthesize MAOM from both adsorption and coprecipitation adds more tunability to the material.

[0153] Natural MAOM is associated with short-range ordered iron oxides and oxyhydroxides12>14>15>32>36Short peptides have been isolated that bind to specific metals and minerals, either by analyzing natural biomineralized bacterial communities or by rational design70-72.

[0154] These peptide sequences can be fused to the genomic copy of tasA in B. subtilis and secreted throughout the biofilm. In addition, as the Bacillus subtilis 168 biofilm is relatively well- characterized, we can knockout the genetic loci for different biofilm polymers and testing the mineral binding affinity of these strains. A summary of the genetic loci and the biopolymers they synthesize is in table 2.

[0155] Without wishing to be bound by theory, Iron (oxyhydr) oxide-binding peptides and biofdm engineering will increase organomineral interactions.

[0156] Engineer organomineral interactions using iron (oxyhydr)oxide-binding peptides'. In embodiments, to facilitate iron (oxyhydr)oxide binding to the biofilm matrix, TasA can be engineered with any one or more of the peptide sequences in table 1. These sequences can be fused to TasA by a flexible Glycine-Serine (GS) linker and integrated into the genomic locus of tasA in B. subtilis 168. Mineral binding affinity can be tested by incubating the engineered biofilm with lab-synthesized ferrihydrite (XRD in figure 5 panel C) and determining the amount of unbound mineral in the supernatant using colorimetric assays.Table 1: Iron (oxyhydr)oxide-binding peptides

[0157] Without wishing to be bound by theory, we can also use a broader peptide screen using SPOT Membrane Arrays77These membranes can be incubated with lab-synthesized 2- line ferrihydrite and binding affinity can be determined by image analysis. High mineral affinity peptides can be secreted using the TasA protein scaffold throughout the biofilm and mineral binding can be determined using colorimetric and spectrometric assays.

[0158] Alternate strategies: Studies on natural biomineralization have highlighted that metal-binding tags act as nucleation sites for mineral crystallization71,78. We can use the metalbinding peptide from natural biomineralization proteins such as ferritin. The peptide sequence is M6A: DIESAQSDEEVE and has been found in multiple biomineralization proteins, as shown in the alignment in figure 4. Another approach involves porting these peptide sequences under strong promoters to plasmid-based systems with gram-positive replication origins. However, the biofilms can be grown without antibiotics to produce reproducible structures. A more prominent design constraint, in this case, is to screen gram-positive replication origins for antibiotic-free persistence across multiple generations of B. subtilis.

[0159] If iron (oxyhydr)oxide-binding peptides do not increase mineral-biofilm interactions due to occlusion by exopolysaccharides and biofilm surface proteins, we can modify organomineral interactions by biofilm engineering. For example, we can use genetic knockouts of different biopolymers produced by B. subtilis to identify the components that increase organomineral interactions. This strategy can inform matrix-level modifications that can be made to the biofilm to increase mineral binding and nucleation. Additionally, this approach will dissect current observations of natural MAOM and determine which chemical moieties are preferentially enriched in MAOM. A summary of strain genotypes and biopolymers produced is in table 2 and the phenotypes of knockout biofilms for major biofilm components is in figure 5 panel G.Table 2: B. subtilis 168 strain genotypes for biofilm knockouts

[0160] To determine the affinity of these modified organic matrices, we can incubate the bacterial cell pellet with Fe2(SO4)3, a soluble Fe3+salt. Samples can be harvested after 24 h, fixed, and imaged using Scanning Electron Microscopy. The supernatant solution from these incubations can be collected and reacted with Ferrozine and Xylenol Orange, two cationbinding dyes, to determine the fraction of Fe mineralized onto the microbial template.

[0161] Without wishing to be bound by theory, we can characterize the persistence of synthetic MAOM.

[0162] The long-term persistence of MAOM in soils is essential for carbon sequestration4>17. We can conduct long-term incubation experiments with the products described herein to characterize the persistence of synthetic MAOM. Synthetic MAOM can be incubated with synthetic and natural soil microbial consortia and degradation rates of engineered MAOM can be measured relative to an unengineered necromass control. We can track the movement of synthetic MAOM using natural isotope ratios (513C) and Stable Isotope Probing with a13C labeled substrate. 513C determines the ratio of13C to12C carbon and is a useful strategy employed for sensitive geochemical measurements that result from isotopic fractionation. Stable Isotope Probing employs labeled substrates to determine the flow of molecules across living systems. These experiments can determine the rates of degradation and temperature sensitivity of the different types of synthetic MAOM engineered as described herein.

[0163] Without wishing to be bound by theory, Mineral-bound organic matter is more resistant to bacterial / microbial degradation than unbound organic matter.

[0164] We can determine the degradation rate of synthetic MAOM by a defined microbial consortium using 513C: We can measure the degradation rate of synthetic MAOM by determining the change in 513C between a defined microbial consortium, such as Mammoth P or a four-species consortium as in McClure et al fed with a maize-based carbon source80,81. Maize is a C4 plant and records a 513C of -12 to -18 %o82. The MAOM can be synthesized with glycerol from a known, petroleum-based source (513C of -22 to -35 %o)82. This difference in 513C will permit a sensitive measurement of the CO2 source. 513C can be measured using a gas isotope analyzer or a GC-IRMS.

[0165] In embodiments, we can determine the degradation rate of synthetic MAOM by a defined microbial consortium using stable isotope probing (SIP): As an alternative to 513C, we can measure the abundance of labeled13CO2 in place of natural abundances. B. subtilis biofilms can be produced with a13C glycerol source. The resulting labeled MAOM can be incubated with a defined microbial community, as described herein. Measurements can be made using a GC-IRMS, as described herein, in a stable isotope facility.

[0166] In embodiments, unlabeled glycerol can be used as a carbon source for biofilm synthesis. The unlabeled glycerol can result isotopically lighter biofilms.

[0167] We can test degradation of synthetic MAOM by natural and defined microbial communities under elevated temperatures: To determine the persistence of synthetic MAOM under elevated temperatures, we can measure Q10, a metric for soil respiration with respect to temperature84We can run bioreactors with natural and defined microbial communities under different temperatures that can be representative of climate change scenarios proposed by the IPCC report. Measurements can be conducted with the additional control of readily processed carbon, such as13C glucose or13C chitin.

[0168] Natural microbial communities are challenging to isolate and have slower metabolic rates than most lab domesticated species. This can increase the time of these incubations.

[0169] References Cited in this Example

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[0256] Synthetic biology for earth system science

[0257] Natural background carbon fluxes are greater than human emissions. Therefore, without wishing to be bound by theory, we can use synthetic biology to increase soil carbon. The “best” soil carbon can be mineral-associated organic matter (MAOM) and14C dates to 1000s to 10,000s years. (Fig. 6 panels A and B).

[0258] A problem is that soil MAOM stocks saturate globally. Reactive mineral surfaces are created as water flows over rocks and slowly reacts creating nm scale instabilities in mineralsthat microbial waste products sorb to which creates mineral-associated organic matter (Fig. 7 panels A and B).

[0259] We can code soil microbes to produce MAOM. For example, we can determine the chemistry of microbial compounds sorbed naturally to minerals then code soil microbes to overexpress those compounds and then grown them in the present of soluble mineral material (Fig. 8). Without wishing to be bound by theory, increasing the soil C pool can create a large natural carbon sink with many ecological and agricultural co-benefits. Reduced irrigation needs improved ecological resilience, improved pest resistance and improved crop yields reduces fire risk. We can create a fast, stable shunt of CO2 out of the atmosphere in a way that can be commodified.

[0260] Non-Limiting Design of biomineralized bacterial biofilms: Dominant chemical constituents - microbial polysaccharides, membranes, biomass. Subject to saturation - decouple MAOM from soil minerals (Fig. 10). We observed signatures of natural MAOM: close association of organic matter and minerals (Fig. 11), presence of amorphous iron oxides (Fig. 12), and enrichment of carboxylates and depletion of O-alkyls (Fig. 13).

[0261] We can alter biofilm chemistry (Fig. 14). Natural isotope fractionation can be used to identify if a culture is eating lab MAOM (Fig. 16). When grown in LB background, the community appears to ignore lab MAOM (Fig. 17 panel A), but when grown in minimal media, the bacterial community eats lab MAOM (Fig. 17 panel B). Without wishing to be bound by theory, we can test when the community starts eating the biofilm / MAOM, perform headspace optimization by normalizing CO2, and study whether biofilm chemistry influences consumption.EXAMPLE 6

[0262] Addressing scalability with synthetic MAOM

[0263] As discussed herein, the presently disclosed knockout strains can produce biofilm variations in laboratory timescales. However, we have had significant challenges in scaling up biofilm mass for high-throughput experiments.

[0264] To assist with scaling up of biofilm mass synthesis, we deleted the pulcherriminic acid biosynthesis cluster, which can result in continually growing B. subtilis biofilms (Amaouteli 2019). Without being bound by theory, this feature of B. subtilis biofilms can be leveraged to synthesize more biofilm mass, which would subsequently result in more LabMAOM / Synthetic MAOM.

[0265] In embodiments, we deleted the cypX-yvmC cluster from B. subtilis using a two-step double-homologous recombination protocol. In the first step, we transformed naturally- competent B. subtilis 168 cells with plasmid pSS172 to facilitate a scarred knockout of cypX- yvmC, with mScarlet-I for screening and ermB for selection on 1 ug / L Erythromycin. Bright pink colonies were inoculated for competent cell preparation as described previously. Naturally competent AcypX-yvmC::mScarlet-I-ermB cells were transformed with plasmid pSS167 to delete the mScarlet-I-ermB scar and produce a scarless genomic knockout. As pSS167 has sfGFP for screening and a temperature-sensitive oriV for plasmid curing, we selected colonies that displayed bright red and green fluorescence, and cured pSS167 on LB plates at 37 °C for 12 h. Colonies were screened for the absence of cypX-yvmC by colony PCR with oligos OSS1259 and oSS1260.

[0266] As shown in Figure 18, the pulcherriminic acid / pulcherrimin pigment was successfully knocked out of Bacillus subtilis 168. Additionally, the apparent biomass of biofilms without pulcherriminic acid is higher than the apparent biomass of biofilms with pulcherriminic acid.

[0267] Further, in certain embodiments, we restricted all biofilm synthesis to 12-well plates (Corning, #3513) to ensure maximal biofilm synthesis.

[0268] Synthesizing varying biofilm chemistries from one strain

[0269] Described herein is an exemplary protocol by which the carb on: nitrogen composition of the presently disclosed biofilms can be tuned with the single tuning knob of glycerol concentration during biofilm synthesis. In certain embodiments, the presently disclosed synthesis strategy resulted in biofilms with varying bulk chemistries that represent a wide range of reduced carbon concentrations (see Figure 19). Thus, in certain embodiments, this single tuning knob enables the synthesis MAOM with different initial biofilm chemistries, while keeping the synthesis protocol and bacterial strain used the same.

[0270] Primers and plasmids used in this example

[0271] Table 5: Primers

[0272] Table 6: Strains

[0273] Table 7: PlasmidsEXAMPLE 7

[0274] Table 9: Primer Sequences

[0275] Table 10 Plasmid ID SequencesEQUIVALENTS

[0276] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

CLAIMSWhat is Claimed is:

1. A method of producing synthetic mineral associated organic matter (MAOM), the method comprising: synthesizing a biofilm by culturing an organism in biofilm-forming conditions, wherein the biofilm produces a biopolymer scaffold; and incubating the biofilm with a mineral or salt, wherein the mineral or salt interacts with the biopolymer scaffold, thereby producing synthetic MAOM.

2. The method of claim 1, wherein the mineral or salt is selected from the group consisting of iron ferric sulfate (Fe2(SO4)3), ferric chloride (FeCh), ferric nitrate (Fe(NOs)3), Ferrihydrite ((Fe3+)2O3 O.5H2O), Lepidocrocite (y-FeO(OH)), Schwertmannite (Fe8O8(OH)e(SO4) • n H2O), or a combination thereof.

3. The method of claim 1, wherein the biofilm-forming conditions comprise a given cell density, lack of agitation, a minimal bio-film promoting media, a given biofilm age, or a combination thereof.

4. The method of claim 1, wherein the biopolymer comprises any one or more of TasA, TapA, exopolysaccharides, y-PGA, or BslA.

5. The method of claim 4, wherein the exopolysaccharide is selected from the group consisting of glucose, N-acetylglucosamine, poly-P-l,6-N-acetyl-D-glucosamine (PNAG), or galactose.

6. The method of claim 1, wherein the biofilm-forming conditions comprise an increased concentration of Mn2+.

7. The method of claim 1, wherein the organism comprises bacterium.

8. The method of claim 7, wherein the bacterium is B. subtilis.

9. The method of claim 8, wherein the B. subtilis is selected from the group consisting ofB. subtilis DK 1042, B. subtilis 3610, or B. subtilis 168.

10. The method of claim 1, further comprising increasing C:N ratio of biofilm by adding glycerol to the organism during culturing.

11. The method of claim 8, wherein the B. subtilis comprises genetically modified B. subtilis.

12. The method of claim 11, wherein the B. subtilis is genetically modified to partially, substantially, or completely delete, silence, inactivate, or down-regulate a gene or fragment thereof that encodes for TasA, TapA, BslA, SipW, EpsA-O, PgsA-E, CypX- YvmC, or any combination thereof.

13. The method of claim 11, wherein the B. subtilis is genetically modified to express one or more iron (oxyhydr)oxide-binding peptides, thereby increasing mineral binding affinity of the biofilm.

14. The method of claim 13, wherein the one or more iron (oxyhydr)oxide-binding peptides) comprises D6, DIO, E6, E5, (TH)n, RRTVKHHVN, or LHHLLHLLHHLLHL.

15. A genetically modified microorganism, wherein the genetically modified microorganism partially, substantially, or completely deleting, silencing, inactivating, or down-regulating a gene or fragment thereof that encodes for TasA, TapA, BslA, SipW, EpsA-O, PgsA-E, CypX-YvmC, or any combination thereof, and thereby is capable of synthesizing a biofilm that produces a biopolymer scaffold, wherein the genetically modified microorganism is genetically modified to express one or more iron (oxyhydr)oxide-binding peptides), thereby increasing mineral binding affinity of the biofilm, or both.

16. The genetically modified organism of claim 15, wherein said microorganism is a bacterium selected from B. subtilis.

17. The genetically modified organism of claim 15, wherein the microorganism comprises AtasA-sipW-tapA, AbslA, AepsA-O, ApgsBCAE, AcypX-yvmC, or any combination thereof.

18. The genetically modified organism of claim 15, wherein the one or more iron (oxyhydr)oxide-binding peptides) comprises D6, DIO, E6, E5, (TH)n, RRTVKHHVN, or LHHLLHLLHHLLHL.

19. A biofilm produced by the method of claim 1 or the genetically modified organism of claim 15.

20. A synthetic MAOM produced by the method of claim 1 or the genetically modified organism of claim 15.

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