Prediction of soil microbial health by measurement of gases formed in the subsoil

Abstract

Described herein are methods of assessing soil microbial health. These methods can comprise measuring a sub-surface concentration of one or more gases selected from the group consisting of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), methane (CH4) or combinations thereof at a depth of from 0.1 inches to 24 inches within a soil sample; and determining the characteristic of soil microbial health from the sub-surface concentration of the one or more gases.

Description

[0001]Attorney Docket No.11632-003WO1 ^ Prediction of Soil Microbial Health by Measurement of Gases Formed in the Subsoil CROSS-REFERENCE TO RELATED APPLICATIONS ^^ This application claims benefit of priority of U.S. Provisional Application No. 63 / 538,468, filed September 14, 2023, which is hereby incorporated herein by reference in its entirety. BACKGROUND ^^^ The plant rhizosphere contains billions of microorganisms per gram of soil. In many cases, these microorganisms are either beneficial or neutral to plant growth. A number of microorganisms are known to be present in soil ecological niche (rhizosphere) having beneficial effects on plant growth. These beneficial plant growth promoting properties can include nitrogen fixation, iron chelation, phosphate solubilization, and the inhibition of non-beneficial or harmful ^^^ microorganisms. Some microorganisms can also improve the resistance to pests and / or can decompose plant material in soil to increase soil organic matter. Soil health in agricultural settings could potentially benefit from a greater understanding of the plant rhizosphere. In particular, a detailed understanding of soil microbial health could improve management decisions and agricultural production. However, to allow for such ^^^ advances, improved methods for assessing and monitoring soil microbial health are needed. SUMMARY Described herein are methods for assessing a characteristic of soil microbial health within a soil sample. In some cases, the soil sample can comprise a soil core collected from a location ^^^ of interest (e.g., an agricultural field). In other cases, the soil sample can comprise soil in situ, for example, within an agricultural field. These methods can comprise measuring a sub-surface concentration of one or more gases selected from the group consisting of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), methane (CH4) or combinations thereof at a depth of ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^soil ^^^ microbial health from the sub-surface concentration of the one or more gases. 1 ^ ^ Attorney Docket No.11632-003WO1 ^ In some embodiments, the method comprises measuring the sub-surface concentration of two or more gases selected from the group consisting of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) at a depth of from 0.1 inches to 24 inches ^^ within a soil sample. In some embodiments, the method comprises measuring the sub-surface concentration of three or more gases selected from the group consisting of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) at a depth of from 0.1 inches to 24 inches within a soil sample. In certain embodiments, the method comprises measuring the sub-surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and ^^^ methane (CH4) at a depth of from 0.1 inches to 24 inches within a soil sample. In some embodiments, the method comprises measuring a fingerprint of the relative sub- surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) at a depth of from 0.1 inches to 24 inches within a soil sample. In some embodiments, the method comprises continuously monitoring the sub-surface ^^^ concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4), or a combination thereof at a depth of from 0.1 inches to 24 inches within a soil sample. Continuously monitoring the sub-surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), methane (CH4), or combinations thereof can comprise repeatedly measuring the sub-surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon ^^^ dioxide (CO2), methane (CH4), or combinations thereof at intervals of 24 hours or less (e.g., 12 hours or less, or 8 hours or less) for a period of at least 7 days (e.g., at least 14 days, at least 30 days, at least 60 days, or over an entire growing season. In some embodiments, these methods can provide information regarding temporal variations in soil microbial health (or due to soil microbial health) within a growing season (or longer). ^^^ In some embodiments, the method can comprise measurement of soil samples (e.g., soil cores) taken from an agricultural field. In other embodiments, the method can comprise measurement of soil in situ within an agricultural field. In some embodiments, soil samples can be taken from (or measurements can be made at) a plurality of locations across an agricultural field. In this way, the method can provide information about the spatial variation of soil microbe 2 ^ ^ Attorney Docket No.11632-003WO1 ^ chemistry across the field, spatial microbial heterogeneity (or homogeneity) across the field, or a combination thereof. In some embodiments, the method further comprises measuring a change in the sub- surface concentration of one or more gases selected from the group consisting of nitric oxides ^^ (NOx), nitrous oxide (N2O), carbon dioxide (CO2), methane (CH4) or combinations thereof in response to a change in environmental conditions, such as a change in temperature, a change in moisture levels in the soil, an addition of a nutrient, fertilizer, or amendment to the soil, physical disturbance of the soil (e.g., tilling, aeration, etc.), planting, crop growth, or a combination thereof. Such methods can be used to assess the impact of the change in environmental ^^^ conditions on soil microbial health over period of time. In some embodiments, the method further comprises measuring the sub-surface concentration of ammonia gas. In some embodiments, the method further comprises measuring the sub-surface concentration of water vapor. ^^^ In some the concentration of the one or more gases are measured at a depth of from 0.5 inches to 24 inches, such as from 0.5 inches to 18 inches, from 0.5 inches to 12 inches, from 1 inch to 24 inches, from 1 inch to 18 inches, from 1 inch to 12 inches, from 3 inches to 24 inches, from 3 inches to 18 inches, or from 3 inches to 12 inches. In some embodiments, measuring the sub-surface concentration of one or more gases ^^^ comprises inserting a probe comprising a sensor for each of the one or more gases into the soil. In some embodiments, the concentration of carbon dioxide is measured using a potentiometric sensor. The potentiometric sensor can comprise a BaCO3-coated Li2CO3 sensing electrode. In some embodiments, the concentration of nitrous oxide is measured using a non- ^^^ dispersive infrared sensor. In some embodiments, the concentration of nitric oxides is measured using a potentiometric sensor. The potentiometric sensor can comprise a WO3sensing electrode and a Pt-loaded zeolite Y (PtY) reference electrode. In some embodiments, the concentration of methane is measured using a non-dispersive ^^^ infrared sensor. 3 ^ ^ Attorney Docket No.11632-003WO1 ^ In some embodiments, determining the characteristic of soil microbial health comprises inputting the sub-surface concentration of the one or more gases into a computer model that correlates the sub-surface concentration of the one or more gases to one or more characteristics of soil microbial health. ^^ In some embodiments, the characteristic of soil microbial health comprises a concentration of microbes present in the soil, species of microbes present in the soil, an assessment of real-time soil health, including microbial activity, nutrient cycling, and soil aerobic or anaerobic status, or any combination thereof. In some embodiments, the method can further comprise utilizing the characteristic of soil ^^^ microbial health to make soil management and / or crop management decisions. Examples of soil management and / or crop management decisions can include the selection of a crop to plant in the soil, selection of a fertilizer, bacterial culture, or other amendment to apply to the soil, or a combination thereof. ^^^ DETAILED DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic illustration of chemical pathways active within the plant rhizosphere. Figure 2 is a photograph of a soil core used in proof-of-principle studies. Figure 3 is a plot showing the evolution of N2O gas from subsurface of an anaerobic soil ^^^ core upon exposure to the atmosphere. The plot includes curves for N2O concentration measured using an FTIR detector and a non-dispersive infrared sensor (NDIR sensor). DETAILED DESCRIPTION Microbial enzyme systems below the earth’s soil surface are the key drivers of Earth’s ^^^ biogeochemical cycles. These microbes are too small and are difficult to directly observe. Currently, sophisticated methods, often including culturing in laboratory conditions, are required to quantify microbes within soil samples. Such methods are expensive and slow, limiting the ability of these methods to inform soil management decisions. Described herein are methods for assessing soil microbial health. These methods can ^^^ assess microbial activity via analysis of the reaction products of the microbial chemistry. In cases 4 ^ ^ Attorney Docket No.11632-003WO1 ^ where the reaction products are small molecule gases, then they evolve within the soil. Measurement of these gases can provide a fingerprint of metabolic activity ongoing within the soil that can be used to determine one or more characteristics of soil microbial health within a soil sample. ^^ Figure 1 is an overall schematic of the soil microbial chemistry. Nitrification and denitrification determine the evolution of the nitrogen oxides within the soil. Nitrification reactions occur when there is source of nitrogen (such as fertilizer N) resulting in the formation of nitrates. Under conditions where the level of oxygen is deficient, the denitrification process will begin, resulting in conversion of nitrates to nitrogen oxides, and eventually to nitrogen. If ^^^ the soil is wet, then diffusion of oxygen will be impeded resulting in situation more favorable for denitrification. Methane and CO2 can be considered as the most reduced and most oxidized species of carbon present in soils, all other organic molecules are intermediates in this redox reaction. Methane producing bacteria known as methanogens in soils only become active in anaerobic, ^^^ highly reducing conditions, this being the reason why in anaerobic soils such as rice paddies are a source of methane. Methane oxidizing bacteria, known as methanothrops function in an aerobic environment. Nitrogenous fertilizers will inhibit methane oxidation. Production of ammonia from fertilizers such as urea is mediated by the urease enzyme. Ammonium salts as fertilizers usually do not lead to NH3 production. Applying urea fertilizers to ^^^ subsurface soils will lead to NH3 production in the subsurface. Ammonia volatilization and thereby loss is a significant economic loss and well as cause of air pollution. As described herein, the gaseous end products of soil biological cycles, including carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), nitric oxides (NO and NO2), and combinations thereof, can serve as measurable analytes that can be quantified to assess the ^^^ microbial health of soil samples. The levels of other relevant gaseous analytes (e.g., ammonia, water, etc.) can also be measured. For example, the levels of one or more of these gases present within a soil surface (e.g., measured by a probe placed within the soil, such as at a depth of from 1 inch to 24 inches) can be correlated to the concentration of microbes in the soil (e.g., by comparison to, for example, a standard curve), allowing the measurement of the levels of one or ^^^ more of these gases to readily provide information regarding the presence of microbes within a 5 ^ ^ Attorney Docket No.11632-003WO1 ^ soil sample (e.g., the concentration of microbes, the species of microbes present, or a combination thereof). This information can be used to assess the microbial health of soil, which can then be used, for example, to inform soil microbial management and / or crop management decisions. In some embodiments, the concentration of multiple gases in the soil can be measured ^^ (e.g., by a probe placed within the soil, such as at a depth of from 1 inch to 24 inches) simultaneously and then correlated to provide information regarding the presence of microbes within a soil sample (e.g., the concentration of microbes, the species of microbes present, or a combination thereof). In certain examples, the concentration of two, three, four, five, or six of CO2, CH4, N2O, NO, NO2, or ammonia gas can be measured within the soil sample, and fed into ^^^ a model that provides information regarding the presence of microbes within a soil sample (e.g., the concentration of microbes, the species of microbes present, or a combination thereof). Besides the microbes, gas emissions can be dependent on other factors, including pH, soil texture, soil mineralogy, temperature, and soil water content, which combined together gives the status of soil health. As shown in Figure 1, the gases above are products of metabolism of carbon ^^^ and nitrogen containing species is the soil. The concentrations of the microbe-initiated gases can be input to model(s) that integrate the data to provide information about the soil microbial health, which in a global sense involves microbial biomass, microbe community composition and group abundance. The model(s) outputs can provide information regarding nutrient cycling and soil fertility. Along with other physical and chemical information, agriculturalists can use the ^^^ microbe-initiated gas emissions to make informed decisions regarding soil management (e.g., the addition of soil amendments), crop selection, etc. In some embodiments, methods can involve real time and continuous measurement of carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O) and nitric oxide (NO and NO2) gases present in the subsoil and real-time modeling of this data to generate a comprehensive picture of ^^^ the soil microbial health. Highly selective and sensitive small-footprint sensors provide information on the gas concentrations. For a certain area under consideration, multiple measurements spread across the land can be used to provide statistically sound data for reliable predictions and / or to assess variations in soil quality, for example, in different regions of a field. In order to make the predictions robust, surface measurements of these gases, along with ^^^ temperature, soil moisture and soil pH are also relevant. Because of the complexity of the soil 6 ^ ^ Attorney Docket No.11632-003WO1 ^ microbiome, the models can employ pattern recognition techniques and inform about the global nature of the microbial health. Model prediction examples include extent of microbial diversity and microbial mass. Based on the model output, active intervention can include microbial addition, suppression or need for soil management practices. ^^ Soil microbes, biological communities and the functions they perform are very complex and dynamic and not easily interpreted for field practices. These methods can provide an opportunity for agriculturalists to obtain information about the microbial community of their fields, and will inform their crop management strategies, including tillage, crop rotation cover cropping, and adding compost, manure, and adding and suppressing microbes. ^^^ In some embodiments, the methods can further comprise amending the soil, for example, by fertilization or the addition of bacterial cultures based on the one or more characteristics of soil microbial health within the soil sample. Companies are marketing engineered microbes that can convert nitrogen form the atmosphere to N-species that can be taken up by the plants. Microbe-based farming offers the ^^^ potential to be more efficient for N uptake as compared to conventional inorganic nitrogen fertilizers, without any negative influence on the crop yield. This would suggest that microbe- based farming will have significantly lower N2O and NOx. This can be assessed, verified, and / quantified by measuring subsurface N2O and NOxover the entire growing period. For example, the claims that fertilizers are more efficient in N uptake by plants can be verified using ^^^ the methods described herein. Some efforts have endeavored to replace synthetic fertilizer with microbial nitrogen fertilizer. Using the methods described herein, the exact amounts of N2O being liberated can be quantified. The impact of these practices can be accurately gauged, potentially allowing farmers to realize a benefit in a carbon credit marketplace for the decreased emissions associated with ^^^ their management strategy. Further, critical decisions on the rates and timing of fertilizer application in agriculture often rely on rough heuristics that don’t account for real-time soil conditions and crop requirements under different environmental conditions. Due to current soil sensing technologies that are costly, inaccurate, and operationally difficult to deploy, a lack real-time data from the ^^^ field, farmers often have to depend on their past experiences and rough guesses to decide how 7 ^ ^ Attorney Docket No.11632-003WO1 ^ much fertilizer to use and when to apply it. The methods described herein can provide data that can help farmers make optimal fertilizer use decisions guided by data. The intention is to improve nitrogen use efficiency (NUE), which is expected to substantially improve farmer profitability and reduce emissions. Transparent to the farmer, the real-time soil health data ^^ gathered by this invention on the various subsurface gases emitted will be modeled to provide the farmer with meaningful suggestions as to optimize fertilizer use. Examples of models that can be used include the extensively studied DeNitrification DeComposition (DNDC) model, which can use the data generated by this invention, along with other climate, soil data to predict farming management practices such as optimized fertilizer / manure application rate. ^^^ The United States is also experiencing a surge in the carbon credits market, opening additional revenue streams for farmers who adopt sustainable practices. This market expansion is fueled by corporate demand and supported by a $300 million investment from the USDA, which aims to improve emissions tracking and make the carbon market more accessible, especially for smaller-scale and traditionally underserved farmers. The market's potential is vast, with an ^^^ estimated value of $10 billion based on the capability to sequester 500 million metric tons of carbon at a rate of $20 per credit. The methods described herein can allow farmers to generate and trade carbon credits, aligning economic incentives with environmental stewardship. Sensors The concentration of gases, including nitric oxides (NOx), nitrous oxide (N2O), carbon ^^^ dioxide (CO2), and methane (CH4) can be measured using a variety of sensors. In some embodiments, one or more sensors can be positioned within a probe that can be used to measure subsurface gas concentrations. Use of sensors provide a major advantage over use of instruments (such as FTIR, gas chromatography, laser spectroscopy) in that sensors require minimal human intervention and can provide real time data over extended periods of time. ^^^ Sensors are also more economic than instruments and can be widely used. Examples of suitable sensors for gases of interest are described below. Carbon Dioxide In some embodiments, CO2concentrations can be measured using a potentiometric sensor with a Li3PO4 electrolyte and a BaCO3-coated Li2CO3 sensing electrode. Such sensors can ^^^ be used to measure CO2 over a wide range of concentrations (100 ppm to 20%) at 500oC with 8 ^ ^ Attorney Docket No.11632-003WO1 ^ minimal interference to humidity. The active element in the sensing electrode for CO2detection is Li2CO3. However, sensors with just Li2CO3 electrodes showed interference from humidity, which was eliminated by use of the BaCO3 layer. Infrared spectroscopy as well as electrode preparations involving heating above the eutectic temperature of BaCO3–Li2CO3suggests that ^^ the BaCO3layer wets the Li2CO3electrode surface, making it more hydrophobic and thereby reducing the interference from humidity. Humidity interference in agricultural applications would be a major impediment. These CO2sensors can be readily miniaturized with footprints of the order of millimeters. Methane ^^^ Example methane sensors can employ non-dispersive infrared (NDIR) technology using a compact pentahedron gas-cell. A paraboloid concentrator, two biconvex lenses and five planar mirrors were used to set up the pentahedron structure. The gas cell is endowed with a 170 mm optical path length with a volume of 19.8 mL. The gas-cell was integrated with a mid-infrared light source and a detector as the optical part of the sensor. Concerning the electrical part, a ^^^ microcontroller was used to generate the driving signal for the IR source, and the signal from the detector was sampled by an analog-to-digital converter. A static volumetric method was employed for the experimental setup, and 20 different concentration CH4samples were prepared to study the sensor’s evaluation, which revealed a 1^ detection limit of 2.96 parts-per-million (ppm) with a 43 s averaging time. ^^^ Nitrous Oxide In some embodiments, the sensor can comprise a non-dispersive infrared sensor for N2O, The principal components of the NDIR sensor are an infrared source, a sample chamber, a light filter, and an infrared detector. IR light is emitted from the infrared source and directed to the sample chamber. The gas in the sample chamber absorbs the IR light at wavelengths specific to ^^^ the gas species. The absorption of IR light by the gas depends on the species concentration according to the Beer-Lambert Law. After passing through the sample chamber, the attenuated IR light passes through a filter at the end of the optical path. Light filters are selected to allow through only the specific wavelengths of light that are absorbed by the gas species of interest. The filtered light is then detected by the infrared detector. Quantitative gas species measurement 9 ^ ^ Attorney Docket No.11632-003WO1 ^ is achieved by back calculating from the IR attenuation the concentration through the Beer- Lambert Law. Sensitivity to N2O down to concentrations of 1 ppm has been demonstrated. Nitric oxide and Nitrogen dioxide (NO+NO2=NOx) In some embodiments, the sensor can comprise potentiometric total NOxsensor that can ^^ measure NOx in the ppb-to-ppm range. The sensor works on the well-studied principle of mixed- potential potentiometric sensing. The sensor uses WO3 sensing electrode and Pt-loaded zeolite Y (PtY) acting as the reference electrode in a planar geometry. WO3has a very poor catalytic reactivity for NOxequilibration, therefore the gas passes through the WO3layer without light filter being modified and then take part in the NOx electrochemistry at the triple-point boundary ^^^ (TPB: WO3 / YSZ / gas). On the other hand, Pt-loaded zeolite Y (Pt-Y) has a very high catalytic reactivity with NOx. The NOxgas passes through the PtY is equilibrated, prior to reaching the TPB, and acts as a suitable reference. The measured signal is due to the electrochemical reaction happening at the sensing electrode. The device uses a Pt-zeolite Y filter separate and ahead of the potentiometric YSZ based sensor. The NOxpassing through the zeolite filter gets equilibrated to ^^^ a particular NO + NO2composition depending on the temperature of the zeolite layer and gives the same response to NO and NO2 or mixtures, thereof. A temperature differential between the filter and the sensor increases the sensitivity. The zeolite catalytic layer also eliminates interference to other oxidizable gases, such as carbon monoxide, ammonia and hydrocarbons which can all be found in soil. By packing the sensors in series, sensitivity is increased. One of ^^^ the inventors (SS) has miniaturized the 9-sensor arrays to dimensions of millimeters, with measurement as low as 10 ppb total NOx. EXAMPLES The invention will be described in greater detail by way of specific examples. The ^^^ following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results. Aspects of these methods (e.g., sensors, reaction times, pH values, types and volumes of soils, depths, etc.) can be varied from trial to trial. 10 ^ ^ Attorney Docket No.11632-003WO1 ^ In a proof-of-principle study, a soil core sample from midwestern United States was obtained (Figure 2) and sealed from atmosphere and kept in the dark. After a period of six months, the soil core sample was retrieved, opened to the atmosphere and N2O emission (ppm) from within the soil core (subsurface depths of 0-10cm) was analyzed by a device that penetrated ^^ within the soil and the gases diffusing through the soil collected and measured by a sensing device (Figure 3). The gas measured was N2O using a NDIR sensor, as described above. The gases were also measured by a FTIR instrument to prove the validity of the sensor. Both the sensor and the instrument follow the evolution of the N2O from within the soil for a period of 13 days. The signals from both measurements appear to parallel each other, indicating that ^^^ inexpensive sensors can track the evolution of subsurface gases from the soil. The evolution profile observed in Figure 3 is an indication of the soil microbial chemistry. There are several possible explanations of the observed patterns. First, since the soil cores were maintained in an anaerobic atmosphere, the denitrification enzymes would be activated, and the immediate evolution of N2O during the first five days could be an indication of gases that were formed ^^^ during storage of the soil core. The soil sample was obtained from cultivated land that has been treated with manure, so nutrients for the microorganisms are still available. Second, spikes in N2O emission are observed on days 7 and 9. Though it is difficult to explain precisely why these spikes are observed, we can make some speculations. By this time period, oxygen and humidity would have made its way into the soil core, resulting in altered enzymatic pathways. In the ^^^ presence of oxygen, the nitrification enzymes would be activated and as the oxygen used up, then denitrification would result in the N2O spikes. So, the spikes could be a balancing act between the nitrification and denitrification process, as the oxygen makes its way into the soil. Once the oxygen profile has been established relative to the atmosphere, the N2O evolution dies down as observed between days 10-13. ^^^ The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended as illustrations of a few aspects of the claims. Any methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the methods in addition to those shown and described herein are intended to fall ^^^ within the scope of the appended claims. Further, while only certain representative compounds, 11 ^ ^ Attorney Docket No.11632-003WO1 ^ components, compositions, and method steps disclosed herein are specifically described, other combinations of the compounds, components, compositions, and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein ^^ or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms ^^^ “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of ^^^ the number of significant digits and ordinary rounding approaches. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. 12 ^ ^

Claims

Attorney Docket No.11632-003WO1 ^ WHAT IS CLAIMED IS:

1. A method for assessing a characteristic of soil microbial health within a soil sample, the method comprising: measuring a sub-surface concentration of one or more gases selected from the group consisting of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), methane (CH4) or combinations thereof at a depth of from 0.1 inches to 24 inches within a soil sample^ and determining the characteristic of soil microbial health from the sub-surface concentration of the one or more gases.

2. The method of claim 1, wherein the method comprises measuring the sub-surface concentration of two or more gases selected from the group consisting of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) at a depth of from 0.1 inches to 24 inches within a soil sample.

3. The method of any one of claims 1-2, wherein the method comprises measuring the sub- surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) at a depth of from 0.1 inches to 24 inches within a soil sample.

4. The method of any one of claims 1-3, wherein the method comprises measuring a fingerprint of the relative sub-surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) at a depth of from 0.1 inches to 24 inches within a soil sample.

5. The method of any of claims 1-4, wherein the method comprises continuously monitoring the sub-surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4), or a combination thereof at a depth of from 0.1 inches to 24 inches within a soil sample. 13 ^ ^Attorney Docket No.11632-003WO1 ^ 6. The method of claim 5, wherein continuously monitoring the sub-surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4), or a combination comprises repeatedly measuring the sub-surface concentration of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), methane (CH4), or combinations thereof at intervals of 24 hours or less for a period of at least 7 days.

7. The method of any one of claims 1-6, wherein the method further comprises measuring a change in the sub-surface concentration of one or more gases selected from the group consisting of nitric oxides (NOx), nitrous oxide (N2O), carbon dioxide (CO2), methane (CH4) or combinations thereof in response to a change in environmental conditions, such as a change in temperature, a change in moisture levels in the soil, an addition of a nutrient, fertilizer, or amendment to the soil, physical disturbance of the soil (e.g., tilling, aeration, etc.), planting, crop growth, or a combination thereof.

8. The method of any one of claims 1-7, wherein the method further comprises measuring the sub-surface concentration of ammonia gas.

9. The method of any one of claims 1-8, wherein the method further comprises measuring the sub-surface concentration of water vapor.

10. The method of any of claims 1-9, wherein measuring the sub-surface concentration of one or more gases comprises inserting a probe comprising a sensor for each of the one or more gases into the soil.

11. The method of any of claims 1-10, wherein the concentration of the one or more gases are measured at a depth of from 0.5 inches to 24 inches, such as from 0.5 inches to 18 inches, from 0.5 inches to 12 inches, from 1 inch to 24 inches, from 1 inch to 18 inches, from 1 inch to 12 inches, from 3 inches to 24 inches, from 3 inches to 18 inches, or from 3 inches to 12 inches. 14 ^ ^Attorney Docket No.11632-003WO1 ^ 12. The method of any one of claims 1-11, wherein the concentration of carbon dioxide is measured using a potentiometric sensor.

13. The method of claim 12, wherein the potentiometric sensor comprises a BaCO3-coated Li2CO3sensing electrode.

14. The method of any one of claims 1-13, wherein the concentration of nitrous oxide is measured using a non-dispersive infrared sensor.

15. The method of any one of claims 1-14, wherein the concentration of nitric oxides is measured using a potentiometric sensor.

16. The method of claim 15, wherein the potentiometric sensor comprises a WO3 sensing electrode and a Pt-loaded zeolite Y (PtY) reference electrode.

17. The method of any one of claims 1-16, wherein the concentration of methane is measured using a non-dispersive infrared sensor.

18. The method of any of claims 1-17, wherein determining the characteristic of soil microbial health comprises inputting the sub-surface concentration of the one or more gases into a computer model that correlates the sub-surface concentration of the one or more gases to one or more characteristics of soil microbial health.

19. The method of any of claims 1-18, wherein the characteristic of soil microbial health comprises a concentration of microbes present in the soil, species of microbes present in the soil, an assessment of real-time soil health, including microbial activity, nutrient cycling, and soil aerobic or anaerobic status, or any combination thereof. 15 ^ ^Attorney Docket No.11632-003WO1 ^ 20. The method of any of claims 1-19, wherein the method further comprises utilizing the characteristic of soil microbial health to make soil management and / or crop management decisions. 16 ^ ^

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