FIELD

[0001] The present disclosure relates to, inter alia, phenotypic screening methods that can be used for the screening of nitrogen fixation activity in bacteria

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63/407,027, filed September 15, 2022, the entire contents of which are hereby incorporated by reference in their entireties

BACKGROUND

[0003] Nitrogen is a critical limiting element for plant grow th and production. It is a major component of many important biomolecules such as chlorophyll, amino acids, ATP and nucleic acids. While nitrogen is an essential nutrient for plant growth and development, it is unavailable in its most prevalent form as atmospheric nitrogen. Rather, plants can only utilize reduced forms of nitrogen, for example, in the form of ammonia and nitrate.

[0004] Fertilizers (e.g, organic manure and inorganic ammonia) are a main source of useable forms of nitrogen for commercial crops, but come at a cost. Manufacturing fertilizer processes account three percent of the world’s carbon emissions. Soil bacteria process ammonia from fertilizers and turn it into nitrates and nitrous oxide, a significant greenhouse gas with 300 times the potency of carbon dioxide. Nitrates produced from fertilizers can be leached into the groundwater or be washed out of the soil surface into washed into rivers, lakes and oceans. Nitrogen in these bodies of water increases the population of microscopic organisms, including toxic cyanobacteria that can poison fish and other aquatic animals.

[0005] One promising solution to the environmental problems associated with fertilizers is the use of non-naturally occurring bacteria with enhanced nitrogen fixation activity. Such bacteria could potentially help high-demand crops grow robustly and produce high yields with a reduced need for fertilizer inputs.

[0006] To obtain such nitrogen fixating bacteria, thousands of candidate bacteria must be screened to identify those with enhanced nitrogen fixation activity. Current screening methods, however, use liquid as a primary growth matrix, which does not allow for assessment of the effects of soil-strain interactions on nitrogen fixation. Screens using medium-scale greenhouses or direct-field screens can be time and cost intensive. Accordingly, there is a need for alternative screening methods that allow for effective and reliable screening of candidate bacteria with nitrogen fixation activity.

SUMMARY

[0007] Provided herein are novel phenotypic screening methods that, in embodiments, can be used for the screening of nitrogen fixation activity in bacteria. Such screening methods utilize a plurality of partitions comprising soil that, in embodiments, allow for cost effective and efficient screening of candidate nitrogen-fixing bacteria suitable for growing food crops. \

[0008] In some aspects, provided herein is a for assessing a phenotype of interest in a plurality of cells comprising two or more cells having different genotypes. The method comprises: a) culturing the plurality of cells in a first medium; b) contacting the plurality of cells with a second medium to form a soil screening assay composition; c) transferring the soil screening assay composition to a plurality of partitions each comprising soil, wherein at least two or more cells with different genotypes are transferred into different partitions; d) incubating the transferred cells in the partitions under conditions that allow for assessment of the phenotype of interest; and e) assessing the phenotype of interest. In some embodiments, the phenotype of interest is nitrogen-fixation activity.

[0009] In some embodiments, the cells are incubated in step d) under a limited oxygen condition. In embodiments, the limited oxygen condition comprises about less than about 10%, less than about 5%, or less than about 1% oxygen. In embodiments, the cells are incubated in the absence of oxygen. In embodiments, the nitrogen-fixation activity is assessed by measuring the ammonia produced by the cells.

[0010] In embodiments, the plurality of cells comprises at least five or more cells having different genotypes, and wherein each of the at least five or more cells are transferred into a different partition in step c). In embodiments, the plurality of cells comprises at least about 10 cells having different genotypes.

[0011] In embodiments, the first medium comprises: one or more carbon sources. In embodiments, the carbon source is selected from: raffinose, sucrose, trehalose, glucose, mannose, fructose, galactose, xylose, glutamine, glycerol, arabinose, amylose, amylopectin, and combinations thereof. In embodiments, the first medium further comprises one or more trace metal ions selected from: molybdenum ions, iron ions, manganese ions, or combinations thereof. [0012] In embodiments, the first medium further comprises a nitrogen source. In embodiments, the nitrogen source is glutamine or yeast nitrogen base.

[0013] In some embodiments, the first medium further comprises one or more organic acids. In embodiments, the one or more organic acids is selected from aconitic acid, malic acid, fumaric acid, and pyruvic acid.

[0014] In embodiments, the second medium comprises one or more carbon sources. In embodiments, the carbon source is amylopectin. In embodiments, the second medium further comprises an enzyme that converts the carbon source into a monosaccharide or disaccharide (e.g., D-glucose). In embodiments, the enzyme is an amylase. In embodiments, the amylase is amyloglucosidase

[0015] In embodiments, the second medium further comprises one or more nitrogen sources. In embodiments, the nitrogen source is selected from: ammonium chloride, yeast nitrogen base, sodium nitrate or a combination thereof.

[0016] In embodiments, the moisture content of the soil in each of the plurality of partitions is about 60%-about 75% after addition of the soil screening assay composition.

[0017] In embodiments, the plurality of cells comprises one or more non-naturally occurring bacterium. In embodiments, the one or more non-naturally occurring bacterium is a modified bacterium from the Kosakonia genus, optionally selected from Kosakonia sacchari and Kosakonia radicincitans . In embodiments, the one or more non-naturally occurring bacterium is a modified bacterium from the Klebsiella genus, optionally selected from K. variicola, K. oxytoca, and K. pneumoniae.

[0018] In embodiments, each of the partitions comprise a volume of less about 1 mL to about 20 mL. In embodiments, each of the partitions is a dish. In embodiments, each of the partitions is a well of a multi-well plate. In embodiments, the plurality of partitions comprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 partitions.

[0019] In embodiments, the incubating step d) occurs for about 24-72 hours. In embodiments, culturing step a) occurs for about 24-72 hours.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 depicts an exemplary embodiment of the subject screening method described herein. DETAILED DESCRIPTION

I. Overview

[0021] Provided herein are novel phenotypic screening methods that allow for the screening of a large number of cells in a cost saving and efficient manner. In embodiments, the subject screening methods can be used for the screening of nitrogen fixation activity in bacteria. As the cells are screened in a plurality of partitions comprising soil, such screens, in embodiments, advantageously allow for assessment of cell-soil interactions in a high throughput manner that is not feasible with current screening methods. In embodiments, bacteria identified from such a screen are used as fertilizer substitute for growing plants, including food crops.

[0022] In embodiments of the subject method, a seed training step is carried out to produce an adequate number of candidate cells with normalized growth prior to screening the candidate cells in a soil screening step. After seed training, the cells are contacted with a soil screening assay medium and the resulting composition is transferred into a plurality of partitions comprising soil. In embodiments, each partition contains only one cell strain. The cells are incubated in the partition for a period of time and the cell strains are screened for the phenotype of interest (e.g., nitrogen-fixation activity).

[0023] Figure 1 depicts an exemplary embodiment of the subject screening method described herein, wherein candidate bacteria are screened for nitrogen fixation activity. In this particular embodiment, seed training occurs for 72 hours. Cells in the seed training culture are then diluted in a soil screening assay medium, and added to soil plate partitions for incubation for 72 hours under hypoxic conditions (1% O2). Following incubation, ammonia titers are measured using a detectable label that binds fixed nitrogen. Aspects of the subject methods are discussed in further detail below.

II. Definitions

[0024] In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.

[0025] The term “a” or “an” refers to one or more of that entity, i.e. can refer to a plural referent. As such, the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0026] “Bacteria” or “eubacteria” refers to a domain of prokaryotic organisms. Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas),' (2) Proteobacteria, e.g., Purple photosynthetic+non- photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia,' (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; and (11) Thermotoga and Thermosipho thermophiles.

[0027] The terms “non-naturally occurring bacterium” refers to a bacterium that has been modified by the cloning and transformation methods of the present disclosure. Thus, the terms include a bacterium that has been altered, modified, or engineered, such that it exhibits an altered, modified, or different genotype and/or phenoty pe (e.g., when the modification affects coding nucleic acid sequences of the bacteria), as compared to the naturally-occurring bacterium from which it was derived. It is understood that in embodiments, the terms refer not only to the particular recombinant bacterium in question, but also to the progeny or potential progeny of such a bacterium.

[0028] The term “wild-type bactenum” describes a bacterium having a genotype that occurs in a natural population of bacteria.

[0029] The term “engineered” or “modified” may refer to any manipulation of a host cell’s genome (e.g. by insertion, deletion, mutation, or replacement of nucleic acids).

[0030] The term “control” or “control host cell” or “control bacterium” refers to an appropriate comparator host cell for determining the effect of a modification or experimental treatment. In embodiments, the control host cell is a wild-type cell. In other embodiments, a control host cell is genetically identical to the modified host cell, save for the modification(s) differentiating the treatment host cell. [0031] As used herein, the term “locus” (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.

[0032] As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism, or group of organisms which results from the interaction between that individual’s genetic makeup (i.e., genotype) and the environment.

[0033] As used herein, the term “chimeric” or “recombinant” when describing a nucleic acid sequence or a protein sequence refers to a nucleic acid, or a protein sequence, that links at least two heterologous polynucleotides, or two heterologous polypeptides, into a single macromolecule, or that re-arranges one or more elements of at least one natural nucleic acid or protein sequence. For example, the term “recombinant” can refer to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0034] As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and/or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like. The terms “nucleic acid” and “nucleotide sequence” are used interchangeably.

[0035] As used herein, the term “gene” refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0036] As used herein, the term “endogenous” or “endogenous gene,” refers to the naturally occurring gene, in the location in which it is naturally found within the host cell genome. An endogenous gene as described herein can include alleles of naturally occurring genes that have been mutated according to any of the methods of the present disclosure. [0037] As used herein, the term “protein modification” refers to, e.g., amino acid substitution, amino acid modification, deletion, and/or insertion, as is well understood in the art.

[0038] Variant polynucleotides also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) PNAS 91 : 10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al.(\997) J. Mol. Biol. 272:336-347; Zhang eU?/.(1997) PNAS 94:4504-4509; Crameri et cz/.(1998) Nature 391:288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458.

[0039] As used herein, “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In embodiments, the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity .

[0040] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0041] It is noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of such exclusive terminology as “solely,” “only,” and the like in connection with the recitation of claim elements or use of a “negative” limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the invention, representative illustrative methods and materials are now described.

[0042] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0043] Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. [0044] Before the invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary . It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherw ise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number, which, in the context presented, provides the substantial equivalent of the specifically recited number.

[0046] All publications, patents, and patent applications cited in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated herein by reference to disclose and describe the subject matter in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the invention described herein is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided might be different from the actual publication dates, which may need to be independently confirmed. III. Candidate cells

[0047] Any suitable cell can be used in the subject screening assay. In embodiments, the method is used to screen for candidate bacteria with enhanced nitrogen-fixing activity. In embodiments, the cells are non-naturally occurring cells with one or more modifications that enhance nitrogen fixation activity. In embodiments, the cells used in the subject methods are modified using a parent wild-type cell. In embodiments, the cells used in the subject methods are modified using a non-naturally occurring parent cell that includes one or more modifications for enhanced nitrogen fixation activity. Exemplary bacterial modifications for enhanced nitrogen fixation activity are disclosed, for example, in US20220127627A1, US20200331820A1, US9975817B2, which are incorporated by reference in pertinent parts relating to nitrogen-fixation enhancing modifications.

[0048] Exemplary bacteria that can be used in the subject methods include diazotrophs that are capable of fixing atmospheric nitrogen gas into a more usable form such as ammonia. Such diazotrophs include, but are not limited to anaerobes (e.g., Clostridium and Desulfovibrio),' facultative anaerobes (Klebsiella pneumoniae, Paenibacillus polymyxa, Bacillus macerans, and Escherichia intermedia,' cyanobacteria; anoxygenic photosynthetic bacteria; Rhizobia, and Frankias. In some embodiments the non-naturally occurring bacteria used in the subject screens are modified Klebsiella. In certain embodiments, the non-naturally occurring bacteria are modified Klebsiella variicola, Klebsiella oxytoca, or Klebsiella pneumoniae. In exemplary embodiments, the non-naturally occurring bacteria are modified Klebsiella variicola.

[0049] In embodiments, at least about 50, at least about 100, at least about 150, at least about 250, at least about 500, at least about 1 x 10 ³ , at least about 2.5 x 10 ³ , at least about 5.0 x 10 ³ , at least about 7.5 x 10 ³ , at least about 1 x 10 ⁴ , at least about 2.5 x 10 ⁴ , at least about 5.0 x 10 ⁴ , at least about 7.5 x 10 ⁴ , or at least about 1.0 x 10 ⁵ different bacterial strains can screened at the same time using the subject methods.

[0050] Modified bacteria for use with the subject screening methods can be made using any suitable method known in the art. Exemplary techniques for modifying microbes, including bacteria, include but are not limited to: polymerase chain reaction (PCR) mutagenesis (error- prone PCR), oligonucleotide-directed mutagenesis, multiplex automated genome engineering (MAGE), PFunkel, homologous recombination including those that utilize programmable nucleases (e.g, ZFNs, TALENS, CRISPR/Cas9, meganucleases), orthogonal DNA polymerase-plasmid pairs, targeting glycosylase to embedded arrays for mutagenesis (TaGTEAM), retrotransposons-based and targeted mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis (DNA shuffling). See also U.S. Pat. No. 8,795,965, 7,132,265, 6,713,285, 6,673,610, 6,391,548, 5,789,166, 5,780,270, 5,354,670, 5,071,743, and US20050266541 and US20100267147, which are incorporated by reference herein, particularly in pertinent parts relating to modification methods.

[0051] In some embodiments, the modifications are introduced in a parent bacterium by homologous recombination mutagenesis. Homologous recombination mutagenesis involves recombination between a DNA fragment that includes replacement nucleic acid of interest and the targeted endogenous polynucleotide sequence. After a double-stranded break occurs, sections of DNA around the 5' ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3' end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. The method can be used, for example, to delete a gene or regulatory element, add a gene or regulatory element, and introduce point mutations. Homologous recombination mutagenesis can be permanent or conditional. Typically, a recombination template is also provided. A recombination template may be a component of another vector, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a site-specific nuclease. A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary' to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g., about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence. Non-limiting examples of site-directed nucleases useful in methods of homologous recombination include zinc finger nucleases, CRISPR nucleases, TALE nucleases, and meganucleases. See, e.g., U.S. Pat. No. 8,795,965 and US20140301990, which are incorporated by reference herein. particularly in pertinent parts relating to homologous recombination mutagenesis techniques. In some embodiments, homologous recombination is performed using a suicide plasmid that includes the replacement nucleic acid of interest and one or more selectable markers.

[0052] Introducing genetic variation may be an incomplete process, such that some bacteria in a treated population of bacteria carry a desired mutation while others do not. In some cases, it is desirable to apply a selection pressure so as to enrich for bacteria carrying a desired genetic variation. Traditionally, selection for successful genetic variants involved selection for or against some functionality imparted or abolished by the genetic variation, such as in the case of inserting antibiotic resistance gene or abolishing a metabolic activity capable of converting a non-lethal compound into a lethal metabolite. It is also possible to apply a selection pressure based on a polynucleotide sequence itself, such that only a desired genetic variation need be introduced (e.g., without also requiring a selectable marker). In this case, the selection pressure can comprise cleaving genomes lacking the genetic variation introduced to a target site, such that selection is effectively directed against the reference sequence into which the genetic variation is sought to be introduced. Typically, cleavage occurs within 100 nucleotides of the target site (e.g., within 75, 50, 25, 10, or fewer nucleotides from the target site, including cleavage at or within the target site). Cleaving may be directed by a site-specific nuclease selected from the group consisting of a Zinc Finger nuclease, a CRISPR nuclease, a TALE nuclease (TALEN), or a meganucleases. Such a process is similar to processes for enhancing homologous recombination at a target site, except that no template for homologous recombination is provided. As a result, bacteria lacking the desired genetic variation are more likely to undergo cleavage that, left unrepaired, results in cell death. Bacteria surviving selection may then be isolated for use in exposing to plants for assessing conferral of an improved trait.

IV. Seed training

[0053] In embodiments of the subject methods, a seed train is performed to produce an adequate number of candidate cells with normalized growth prior to screening in the soil containing partitions (i.e., the soil screening step). In embodiments, the seed training step comprises culturing bacteria used from the soil screen in a seed training culture medium that is a nutrient rich medium. An exemplary seed training culture medium for use with the subject methods described herein is shown in Table 1 (see Examples). [0054] In some embodiments, the seed training culture medium includes one or more carbon sources. Exemplary carbon sources that are useful with the seed training culture medium include but are not limited to: raffinose, sucrose, trehalose, glucose, mannose, fructose, galactose, xylose, glutamine, glycerol, arabinose, amylose, amylopectin, and combinations thereof In embodiments, the seed culture medium includes D-raffinose, sucrose, D-trehalose, D-glucose, D-mannose, D-fructose, D-galactose, L-arabinose, D-xylose, and/or L-glutamine, or combinations thereof. In some embodiments, the seed training culture medium includes about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 different carbon sources. In some embodiments, the seed training culture medium includes only glucose as the carbon source. In embodiments, the seed training culture medium includes only amylopectin as the carbon source. In embodiments, each carbon source is present in the seed training culture medium at a concentration of about 1 mM to about 10 mM, about 5 mM to about 15 mM, about 10 mM to 20 mM, about 15 mM to 25 mM, about 20 mM to about 30 mM, about 25 mM to about 35 mM, about 30 mM to about 40 mM, about 35 mM to about 45 mM, or about 40 mM to about 50 mM. In exemplary embodiments, each carbon source is present in the seed training culture medium at a concentration of about 10 mM to 15 mM.

[0055] In some embodiments, the seed training culture medium includes a nitrogen source. Exemplary suitable nitrogen sources include, but are not limited to include anhydrous ammonia, ammonia sulfate, urea, diammonium phosphate, urea-form, monoammonium phosphate, ammonium nitrate, nitrogen solutions, calcium nitrate, potassium nitrate, sodium nitrate, glutamine, and yeast nitrogen base (without ammino acids or ammonium sulfate). In some embodiments, the seed training culture medium includes glutamine and/or yeast nitrogen base (without ammino acids or ammonium sulfate).

[0056] In some embodiments, the seed training culture medium includes one or more organic acids. Suitable organic salts for use in the seed training culture medium include, but are not limited to, aconitic acid, malic acid, fumaric acid, pyruvic acid and combinations thereof. In some embodiments, the seed training culture medium includes about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 different organic salts. [0057] In some embodiments, the seed training culture medium further includes one or more buffering agents. Exemplary buffering agents that find use in the seed training culture medium include, but are not limited to sodium citrate, ascorbate, succinate, lactate, citric acid, boric acid, borax, hydrochloric acid, disodium hydrogen phosphate, acetic acid, formic acid, glycine, bicarbonate, phosphate, tartaric acid, Tris-glycine, Tris-NaCl, Trisethylenediamine tetraacetic acid (“EDTA”), Tris-borate, Tris-borate-EDTA, Tris-acetate- EDTA (“TAB”), Tris- buffered saline, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (“HEPES”), 3-(N- morpholino) propanesulfonic acid (“MOPS”), piperazine- l,4-bis(2- ethanesulfonic acid) (“PIPES”), 2-(N-morpholino)ethanesulfonic acid (“MES”), potassium phosphate monobasic, potassium phosphate dibasic, and phosphate buffered saline (“PBS”).

[0058] In some embodiments, the seed training culture medium is supplemented with trace metal ions, such as molybdenum ions, iron ions, manganese ions, or combinations of these ions. Exemplary metal ions include, but are not limited to iron (II) sulfate heptahydrate and sodium molybdate dihydrate. In embodiments, the metal ions included the seed training culture medium facilitate nitrogen fixation by functioning as cofactors for the nitrogenase enzyme complex.

[0059] In embodiments, the cells are cultured in the seed training culture medium for about 1 hour, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours. In some embodiments, the cells are culture in the seed training culture medium from about 0- about 12 hours, about 6- about 18 hours, about 12- about 24 hours, about 18- about 30 hours, about 24- about 36 hours, about 30- about 42 hours, about 36- about 48 hours, about 42 about 54 hours, about 48 to about 60 hours, about 54-about 66 hours, about 60 hours-72 hours, about 66 hours-about 78 hours, about 72 hours- 84 hours, about 78 hours-90 hours, or about 84 hours-96 hours. In exemplary embodiments, the cells are cultured for 18-30 hours.

[0060] In some embodiments, the cells are cultured in the seed training culture medium at about 20°C, 25°C, at about 30°C, at about 35°C, at about 45°C, at about 50°C. at about 55°C, at about 60°C, at about 65°C, at about 70°C, at about 75°C, at about or about 80°C. In embodimetns, the cells are cultured in the seed training culture medium at about 20°C- about 30°C, at about 25°C-35°C, at about 30°C-about 40°C, at about 35°C- about 45°C, at about 40°C- about 50°C, at about 45°C- about 55°C, at about 50°C- about 60°C, at about 55°C- about 65°C, at about 65°C- about 75°C, or at about 70°C- about 80°C. In exemplary embodiments, the cells are cultured at 25°C-35°C.

[0061] In some embodiments, the cells are grown the seed training culture medium for at least about 18-30 hours at a temperature of about 25°C-35°C. Following seed training, the cells are contacted with a soil screening assay medium, and transferred to a plurality of partitions comprising soil, wherein the soil screening assay is performed.

V. Soil screening assay

[0062] In embodiments, candidate cells (e.g, bacteria) that will be screened in the soil screening assay are first diluted in a soil screening assay medium In embodiments, the screening assay medium includes one or more nutrients and/or components that facilitate nitrogen fixation (e.g., carbon and nitrogen sources). In embodiments, candidate cells in the soil screening assay medium are then introduced into partitions comprising soil and the soil screening assay is carried to assess one or more phenotypic traits of interest.

A. Soil screening assay medium

[0063] In embodiments, cell strains from the seed training step are diluted a soil screening assay medium prior to adding to soils in partitions. An exemplary soil screening assay medium for use with the subject methods is depicted in Table 2 (see Examples). In embodiments, cells in the seed training culture medium after the seed training step are diluted into the soil screening assay medium at a volume to volume ratio of at least about 1: 1, about 1 :5, about 1: 10, about 1 :15, about 1:20, about 1 :30, about 1:40, about 1:50, about 1:70, about 1 :80, about 1:90, about 1: 100.

[0064] In some embodiments, the soil screening assay medium includes a nitrogen source. Exemplary suitable nitrogen sources include, but are not limited to include anhydrous ammonia, ammonia sulfate, urea, diammonium phosphate, urea-form, monoammonium phosphate, ammonium nitrate, nitrogen solutions, calcium nitrate, potassium nitrate, sodium nitrate, glutamine, and yeast nitrogen base (without ammino acids or ammonium sulfate). In some embodiments, the soil screening assay medium includes yeast nitrogen base (without ammino acids or ammonium sulfate), ammonium chloride, and/or sodium nitrate.

[0065] In some embodiments, the soil screening assay medium includes one or more carbon sources. Exemplary carbon sources include, but are not limited to: raffinose, sucrose, trehalose, glucose, mannose, fructose, galactose, xylose, glutamine, glycerol, arabinose, amylose, amylopectin and combinations thereof. In embodiments, the carbon source is amylose or amylopectin. In embodiments, the carbon source is amylopectin. In some embodiments, the soil screening assay medium only includes amylopectin as a carbon source.

[0066] In exemplary embodiments, the soil screening assay medium include one or more enzymes that allows for the slow release of glucose in the soil during the assay. In embodiments wherein the candidate cells are unable to metabolize the carbon source in included in the soil screening assay medium directly, the soil screening assay medium include an enzyme that converts the carbon source into glucose monomers that the candidate cells can metabolize. In some embodiments, the soil screening assay medium includes an amylase. In some embodiments, the amylase is a y-amylase. Such y-amylases are capable of cleaving a(l-6) glycosidic linkages, as well as the last a-1,4 glycosidic bond at the nonreducing end of amylose and amylopectin, to yield D-glucose monomers. In exemplary embodiments, the amylase is amyloglucosidase. In exemplary embodiments, the amyloglucosidase. is present in the soil screening assay medium is a concentration of about less than 1 U/ml. In some embodiments, the amyloglucosidase is present in the soil screening assay medium at about less than 1 U/ml , about less than 0.9 U/ml, about less than 0.8 U/ml, about less than 0.7 U/ml, about less than 0.6 U/ml, about less than 0.5 U/ml, about less than 0.4 U/ml, about less than 0.3 U/ml, about less than 0.2 U/ml, or about less than 0.1 U/ml. In embodiments, the slow release of glucose monomers advantageously allows for the controlled growth of candidate cells that metabolize glucose. In embodiments, controlled release of glucose mimics the release of carbon source from roots in the field soil (e g., com roots).

[0067] In some embodiments, the soil screening assay medium includes a buffering agents that provides consistency in the pH during the course of screening in the soil screening assay. In some embodiments, the buffering agent provides consistency in the pH of the soil screening assay throughout the course of phenotypic assessment. In some embodiments, the buffering agent provides consistency in the pH during the course of the soil assay for at least about 1 hour, at least about 2 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about one week, at least about 2 weeks, at least about one month, at least about, two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, at least about twelve months, at least about eighteen months, or at least about two years. In some embodimetns, the buffer agent maintains the pH of the soil during the soil screening assay at a pH range of about 5-9, about pH 5-8, about pH 5-7, about pH 5-6, about pH 6-9, about pH 6-8, about pH 6-7, about pH 7-9, or about pH 7-8. In some embodiments, the buffering agent maintains the pH of the soil during the soil screening assay in the pH range of pH 6-8. In embodiments, the buffering agent maintains the pH of the soil screening assay at a pH similar to that of soil at a particular geographical region of interest. In embodiments, the pH range is from about pH 6.0-6.5. Non-limiting examples of buffering agents suitable for use within the disclosed soil screening assay medium include sodium citrate, ascorbate, succinate, lactate, citric acid, boric acid, borax, hydrochloric acid, disodium hydrogen phosphate, acetic acid, formic acid, glycine, bicarbonate, phosphate, tartaric acid, Tris-glycine, Tris-NaCl, Tris-ethylenediamine tetraacetic acid (“EDTA”), Tns-borate, Tns-borate-EDTA, Tns-acteate-EDTA (“TAB”), Tris- buffered saline, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (“HEPES”), 3-(N- morpholino) propanesulfonic acid (“MOPS”), piperazine- l,4-bis(2-ethanesulfonic acid) (“PIPES”), 2-(N-morpholino)ethanesulfonic acid (“MES”), potassium phosphate monobasic, potassium phosphate dibasic, and phosphate buffered saline (“PBS”).

[0068] In some embodiments, the soil screening assay medium is supplemented with trace metal ions, such as molybdenum ions, iron ions, manganese ions, or combinations of these ions. Sources of metal ions that can be included in the soil screening assay medium, but are not limited to iron (II) sulfate heptahydrate and sodium molybdate dihydrate. In some embodiments, the trace metal ion is present in the soil screening assay medium at a concentration of about 1 pM to 50 pM, about 5 pM to 30 pM, or about 10 pM to 20 pM. In embodiments, the metal ions included the soil screening assay medium facilitate nitrogen fixation by functioning as cofactors for the nitrogenase enzyme complex.

[0069] In embodiments, after contacting the cells with the soil screening assay medium, the cell in soil medium are added to individual partitions that comprise dry soil. In some embodiments, the soil screening assay medium that includes the candidate cells are added to dry soil at a ratio of about 0.25 ml, about 0.50 ml, about 0.75 ml, about 1.0 ml, about 1.25 ml, about 1 .50 ml, about 1 .75 ml, about 2.0 ml, about 2.25 ml, about 2.50 ml, about 2.75 ml, or about 3.0 ml of soil screening assay medium to about 1.0 g of dry soil. In some embodiments, the soil screening assay medium that includes the candidate cells are added to dry soil at a ratio of about 0.25 ml-about 0.75 ml, about 0.50 ml to about 1.0 ml, about 0.75 ml to about 1.25 ml, about 1.0 ml to about 1.50 ml, about 1.25 ml to about 1.75 ml, about 1.50 ml to about 2.0 ml, about 1.75 ml to about 2.25 ml, about 2.0 ml to about 2.50 ml, or about 2.25 ml to about 2.75 ml of soil screening assay medium to about 1.0 g of dry soil. In some embodiments, the soil screening assay medium that includes the candidate cells are added to dry soil at a ratio of about 0.50 ml-about 1.0 ml, about 1.0 ml-about 1.50 ml, about 1.50 ml- about 2.0 ml, about 2.0 ml-about 2.50 ml, or about 2.50 ml- about 3.0 ml of soil screening assay medium to about 1.0 g of dry soil. In exemplary embodiments, the soil screening assay medium that includes the candidate cells are added to dry soil at a ratio of about 1.5 ml-2.0 ml of soil screening assay medium to about 1.0 g of dry soil.

[0070] As used herein “dry soil” refers to a dry soil mix that includes less than 10% water, less than 5% water, less than 2.5% water, or less than 1.0% water by weight. Dry soil can be obtained by any suitable method known in the art. In some embodiments, dry soil is obtained by baking soil at high temperatures (e.g., above 60°C) to remove water.

[0071] In embodiments, the soil used for the soil screening assay includes peat, processed forest products, coir, compost, and/or sphagnum peat moss. In some embodiments, the total amount of the nitrogen in the dry soil used is no more than about 5%, no more than about 2.5%, no more than about 1%, no more than about 0.5%, no more than about 0.1%, no more than about 0.05% of the dry soil. In some embodiments, the total amount of phosphate in the dry soil is no more than about 5%, no more than about 2.5%, no more than about 1%, no more than about 0.5%, no more than about 0.1%, no more than about 0.05% of the dry soil. In some embodiments, the total amount of soluble potash in the dry soil is no more than about 5%, no more than about 2.5%, no more than about 1%, no more than about 0.5%, no more than about 0.1%, no more than about 0.05% of the dry soil. In some embodiments, the dry soil further comprises sand and/or clay.

[0072] In embodiments, cells are added to the soil screening assay medium at a suitable concentration to assess for phenotype. In some embodiments, the cells are present in each partition at a concentration of at least about 100, at least about 200, at least about 500, at least about 1 x 10 ³ , at least about 2.5 x 10 ³ , at least about 5.0 x 10 ³ , at least about 7.5 x 10 ³ , at least about 1 x 10 ⁴ , at least about 2.5 x 10 ⁴ , at least about 5.0 x 10 ⁴ , at least about 7.5 x 10 ⁴ , at least about 1 x 10 ⁵ , at least about 2.5 x 10 ⁵ , at least about 5.0 x 10 ⁵ , at least about 7.5 x 10 ⁵ , at least about 1 x 10 ⁶ , at least about 2.5 x 10 ⁶ , at least about 5.0 x 10 ⁶ , at least about 7.5 x 10 ⁶ , at least about 1 x 10 ⁷ , at least about 2.5 x 10 ⁷ , at least about 5.0 x 10 ⁷ , at least about 7.5 x IO ⁷ , or at least about 1 x IO ⁸ cells per partition. In embodiments, the cells are present in each partition at a concentration of at least about 1 x 10 ⁵ CFU/mL, at least about 5 x 10’ CFU/mL, at least about 1 x 10 ⁶ CFU/mL, at least about 5 x 10 ⁶ CFU/mL, at least about 1 x 10 ⁷ CFU/mL, at least about 5 x 10 ⁷ CFU/mL, at least about 1 x 10 ⁸ CFU/mL, at least about 5 x 10 ⁸ CFU/mL, at least about 1 x 10 ⁹ CFU/mL, at least about 5 x 10 ⁹ CFU/mL, at least about 1 x IO ¹⁰ CFU/mL, or at least about 5 x IO ¹⁰ CFU/mL.

[0073] In some embodiments, the soil screening assay medium with the cells contacted with the dry soil and then portion of the resulting mixture is added to one or more partitions. In some embodiments, the dry soil is first added to the partitions and aliquots of the soil screening assay medium with the cells are added to the partitions comprising the dry soil.

[0074] Any suitable partition can be used for the subject phenotypic screening methods provided herein. In embodiments, the screening methods provided herein allows for large scale screening of a plurality of non-naturally occurring cells in a space-saving and cost- effective manner. Thus, in embodiments, the screening methods provided herein are carried out in a plurality of partitions. In embodiments, each of the partition includes a plurality cells with the same genoty pe. In embodiments, each of the partition includes a plurality bacteria of the same bacterial strain. In embodiments, each of the partitions do not include cells of more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different genotypes. In embodiments, each partition includes cells of one genotype. In embodiments, each partition includes only one bacterial strain. In some embodiments, the screening method is carried out using at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 250, at least about 500, at least about 750, least about 1 x 10 ³ , at least about 2.5 x 10 ³ , at least about 5.0 x 10 ³ , at least about 7.5 x 10 ³ , at least about 1 x 10 ⁴ , at least about 2.5 x 10 ⁴ , at least about 5.0 x 10 ⁴ , at least about 7.5 x 10 ⁴ , or at least about 1 x 10 ⁵ different partitions, wherein each partition includes cells with a different genotype. Exemplary' partitions include, not are not limited to, wells, plates, test tubes, vials, flasks, bottles, ampules, syringes, or the like. In some embodiments, the screening methods are carried out in multi-well plates that comprise at least 6, 12, 24, 48, or 96 wells in each plate. [0075] In embodiments, the partitions are less than 1,000 mL in volume. In some embodiments, the partitions are less than about 900 ml, less than about 800 ml, less than about 700 ml, less than about 600 ml, less than about 500 ml, less than about 400 ml, less than about 300 ml, less than about 250 ml, less than about 200 ml, less than about 150 ml, less than about 100 ml, less than about 90 ml, less than about 80 ml, less than about 70 ml, less than about 60 ml, less than about 50 ml, less than about 40 ml, less than about 30 ml, less than about 20 ml, less than about 10 ml, or less than about 5 ml.

B. Phenotypic assessment

[0076] Cells are maintained in the soil containing partitions under particular suitable conditions, depending on the phenotype assessed. In embodiments, the methods are used to assess nitrogen fixation activity of candidate non-naturally occurring bacteria. In such embodiments, the cells are incubated in the partitions for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours prior to assessment of nitrogen fixation activity.

[0077] In some embodiments, the screening methods are used to screen for candidate bacteria that are capable of fixating nitrogen under anaerobic (0% oxygen) or hypoxic (2% or less oxygen) conditions. Thus, in some embodiments, the screening methods are carried out in an anaerobic incubator or an incubator that allows for reduced oxygen conditions (e.g., about 20%, about 15%, about 10%, about 5%, about 2%, about 1%, about 0.5%, or less oxygen).

[0078] Nitrogen fixation activity can be assessed using any suitable technique. Exemplary methods for assessing nitrogen fixation activity include, but are not limited to, ¹⁵ N incorporation assays (see, e.g., Chalk, Symbiosis 69:63-80 (2016), Doty et al., Symbiosis 39:27-35 (2016), Herridge and Giller, Working with Rhizobia, eds J. G. Howieson and M. J. Dilworth, pp. 187-218 (2016), Van Deynze et al. PLoS Biol. 16:e2006352 (2018)), and acetylene reduction assays (ARAs) (see, e.g., Hardy et al., Plant Physiol. 43: 1185-1207 (1968)). In some embodiments, nitrogen fixation is detected using o-Phthalaldehyde, a fluorescent label capable of detecting nitrogen fixed as ammonia (see, e.g., Barney et al., Appl Environ Microbiol. 83(2):e01534-17 (2017); and Plunkett et al. Microb Cell Fact. 19: 107 (2020)). [0079] In some embodiments, the biomass of the candidate bacteria is assessed. Methods for assessing microbial biomass in soil are disclosed, for example, in Jenkinson and Ladd, Soil Biochemistry pp. 415-471 (1981); and Martens et al., Biol. Fertil. Soils 19:87-99 (1995).

[0080] In embodiments, the cells are incubated at a particular temperature for phenotypic assessment. In some embodiments, the cells are incubated in the partitions containing soil at temperature of about 50°C, about 55°C about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C. In some embodiments, the cells are incubated in the partitions at a temperature of about 50°C-about 60°C, about 55°C- about 65°C, about 70°C-about 80°C, about 75°C-about 85°C, about 80°C-about 90°C, about 85°C-about 95°C, or about 90°C-about 100°C.

[0081] In embodiments, candidate cells are selected based on a change in phenotype as compared to a control cell. In some embodiments wherein nitrogen fixation activity is assessed in a plurality of candidate bacteria, candidate bacteria are selected for based on enhanced nitrogen fixation activity' as compared to a control bacterium (e.g., a wild-type bacterium). In some embodiments, the candidate bacteria are non-naturally occurring bacteria that include one or more modifications and the control bacterium is a bacterium without the modification. In embodiments, wherein nitrogen fixation activity is assessed, a candidate bacterium is selected if such bacterium exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, or about at least about 200% more nitrogen fixation activity as compared to a control bacterium.

[0082] In embodiments, where an increase in biomass is assessed, a candidate bacterium is selected for use in the subject method if it exhibits at least about a 5% increase, at least about a 10% increase, at least about a 15% increase, at least about a 20% increase, at least about a 25% increase, at least about a 30% increase, at least about a 35% increase, at least about a

40% increase, at least about a 45% increase, at least about a 50% increase, at least about a

55% increase, at least about a 60% increase, at least about a 65% increase, at least about a

70% increase, at least about a 75% increase, at least about a 80% increase, at least about a

85% increase, at least about a 90% increase, at least about a 95% increase, at least about a 100% increase, at least about a 125% increase, at least about a 150% increase, at least about a 175% increase, or about at least about a 200% increase in biomass as compared to a control bacterium.

VI. Preselection

[0083] In some embodiments, candidate cells are preselected using one or more preselection assays prior to use in the subject screening methods.

[0084] In some embodiments, the subject methods are for use in screening non-naturally occurring bacteria with enhanced nitrogen fixation activity. In some embodiments, the bacteria are preselected for enhanced nitrogen fixation activity and/or increase in biomass under hypoxic or aerobic conditions using one or more different screening assays.

[0085] In some embodiments, a candidate bacterium is preselected for use in the subject screening methods if it exhibits an increase in nitrogen fixation activity as compared to a control under limited oxygen conditions of about 20%, about 15%, about 10%, about 5%, about 2%, about 1%, about 0.5%, or less oxygen. In embodiments, a candidate bacterium is preselected for use in the subject method if it exhibits at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, or about at least about 200% more nitrogen fixation activity as compared to a control bacterium.

[0086] In some embodiments, a candidate bacterium is preselected for use in the subject screening methods if it exhibits an increase in biomass as compared to a control under limited oxygen conditions of about 20%, about 15%, about 10%, about 5%, about 2%, about 1%, about 0.5%, or less oxygen. In embodiments, a candidate bacterium is preselected for use in the subject method if it exhibits at least about a 5% increase, at least about a 10% increase, at least about a 15% increase, at least about a 20% increase, at least about a 25% increase, at least about a 30% increase, at least about a 35% increase, at least about a 40% increase, at least about a 45% increase, at least about a 50% increase, at least about a 55% increase, at least about a 60% increase, at least about a 65% increase, at least about a 70% increase, at least about a 75% increase, at least about a 80% increase, at least about a 85% increase, at least about a 90% increase, at least about a 95% increase, at least about a 100% increase, at least about a 125% increase, at least about a 150% increase, at least about a 175% increase, or about at least about a 200% increase in biomass as compared to a control bacterium under limited oxygen conditions. Bacteria biomass can be measured using any suitable technique. In some embodiments, biomass increase is determined based on optical density using a spectrometer.

[0087] some embodiments, a candidate bacterium is preselected for use in the subject screening methods if it maintains a threshold percentage of biomass as compared to a control under limited oxygen conditions of about 20%, about 15%, about 10%, about 5%, about 2%, about 1%, about 0.5%, or less oxygen. In some embodiments, the candidate bacterium is preselected for use in the subject screening methods if it maintains at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% biomass as compared to a control bacterium under limited oxygen conditions.

[0088] In some embodiments, a candidate bacterium is preselected for use in the subject screening methods if it exhibits an increase in nitrogen fixation activity as compared to a control under limited oxygen conditions in the presence of one or more carbon sources. Carbon sources that can be included in the preselection assays include, but are not limited to: raffinose, sucrose, trehalose, glucose, mannose, fructose, galactose, xylose, glutamine, glycerol, arabinose, amylose, amylopectin and combinations thereof. In embodiments, the carbon source is amylose or amylopectin. In embodiments, the carbon source is amylopectin. In some embodiments, the preselection assay only includes amylopectin as a carbon source. In some embodiments, the preselection assay includes D-raffinose, sucrose, D-trehalose, D- glucose, D-mannose, D-fructose, D-galactose, L-arabinose, D-xylose, L-glutamine, and/or combinations thereof. In some embodiments, the preselection assay includes about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 different carbon sources. In embodiments, the carbon source is provided in the preselection assay in a batch or fed-batch manner. [0089] In some embodiments, the preselection assay is carried out in a culture medium that includes one or more organic acids. Suitable organic salts include, but are not limited to, aconitic acid, malic acid, fumaric acid, pyruvic acid and combinations thereof.

[0090] In some embodiments, the preselection assay is carried out for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours prior to phenotypic assessment (e.g., endpoint ammonia titer and/or biomass measurement) and preselection of candidate bacteria.

[0091] In some embodiments, a bacteria strain is preselected for the subject screening methods provided using a desirability score. In embodiments, desirability is a mathematical function to assign each strain a single value (“desirability score” or “score”) ranging from 0 (least desirable) to 1 (most desirability). In embodiments, each response (e.g. biomass, ammonia titer) receives a desirability score, then all of the scores are combined, weighted, and averaged (geometric mean) to generate an overall score. n some embodiments, a desirability score is based on biomass and nitrogen fixation. In some embodiments, biomass and nitrogen fixation (e.g., ammonia titer) are weighted equally when determining a desirability score. In embodiments, nitrogen fixation (e.g, ammonia titer) is given a greater weight than biomass when determining a desirability score. In some embodiments, biomass is given a greater weight than nitrogen fixation (e.g., ammonia titer) when determining a desirability score.

EXAMPLES

[0092] Examples are provided below to illustrate the present invention. These examples are not meant to constrain the present invention to any particular application or theory of operation.

Example 1 - Seed training and soil screening assay

[0093] Candidate cell strains were first grown from frozen glycerol stock into generic nutrient rich medium, “super optimal broth” or “SOB.” See, e.g., Hanahan, J. Mol. Biol. 166(4):557-80 (1983). Cells are grown for 24 h at 30°C. Cells were transferred to the medium shown in Table 1.

Table 1: Exemplary bacteria seed training culture medium

[0094] Cell strains were grown for 24 h at 30°C, and then added to the soil screening assay medium shown in Table 2 at a 1 : 10 dilution.

Table 2: Soil Assay Medium

[0095] For each cell strain, a 1.8 ml aliquot of the cell strain in soil screening assay medium was added to a well of a 24-well plate that contains 1g of soil. After addition of the cell strain in soil screening assay medium to the soil, the final soil moisture content was 64.3% (assuming 1 ml of medium is equivalent to 1 g of medium), which is typical for com fields at planting. The 24-well plates are then incubated in 1% O2 in an anaerobic chamber for 72 h without shaking. Endpoint ammonia titer was extracted from the soil then measured.

Ammonia titer for each of the samples was measured using an o-phthalaldehyde (OPA)-based assay. [0096] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains.

[0097] All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein.

[0098] All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0099] Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.