Inhibition of microbial methanogenesis
By modifying the hypophosphite oxidase gene in crops such as rice, and using hypophosphite as an inhibitor and phosphorus source, the shortcomings of existing microbial methanogenesis inhibitors have been overcome, achieving efficient and economical methane inhibition and deep carbon sequestration.
Patent Information
- Application Number
- CN202480042018.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-06-23
- Filing Date
- 2024-06-23
- Publication Date
- 2026-01-23
AI Technical Summary
Existing inhibitors of microbial methanogenesis are limited in number, non-specific, and require high concentrations to be effective. They are not feasible for application in many environmental systems, and existing studies have not considered the efficacy or specificity of hypophosphite as an inhibitor of methanogenesis.
By using hypophosphite as a formate analogue, the htxA gene of hypophosphite/2-oxoglutarate dioxygenase was expressed by modifying plant genes. This allows the plant to act as a phosphorus source in crops such as rice, while inhibiting the syntrophic methanogenesis of complex environmental microbial communities. It also serves as a more mobile phosphate fertilizer in the soil for deep soil carbon sequestration.
It achieves selective inhibition of methanogenesis in complex microbial communities, improves the efficiency of phosphate fertilizer amendments in farmland, reduces methane emissions, and increases carbon storage in deep soil. Furthermore, hypophosphite is stable and effective in anaerobic environments, resulting in significant cost-effectiveness.
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Abstract
Description
[0001] introduction
[0002] Microbial methanogenesis is a major source of methane emissions into the atmosphere (Rosentreter et al., 2021). However, known inhibitors of microbial methanogenesis are limited in number, non-specific, and require high concentrations to be effective (Liu et al., 2011). Nevertheless, the methanogenesis pathway is highly conserved and specific to methanogens, thus possessing significant potential for drug development. Commonly used methanogenesis inhibitors include compounds such as bromoethanesulfonic acid (BES), 3-nitrooxypropanol (3-NOP), lumazine, and halogenated analogs of methane or acetate (Liu et al., 2011). These compounds exhibit varying inhibitory efficacies, are costly to produce, and are not feasible for use in many environmental methanogenesis systems.
[0003] Microbial methanogenesis in an environmental system involves a synergistic degradation process of complex organic carbon by different bacterial subgroups. These subgroups sequentially depolymerize the complex organic carbon, followed by fermentation to generate organic acids. Subsequently, the synergistic bacteria convert these organic acids into hydrogen, formate, and acetate, which serve as the primary electron donors for methanogenic archaea. Figure 1 When the concentrations of hydrogen, formate, and acetate are maintained at low levels by the activity of methanogens, the symbiotic transformation of organic acids is thermodynamically favorable. Therefore, it has been found that in natural ecosystems of methane production, symbiotic bacteria and methanogenic archaea are closely associated, and inhibiting either group inhibits methane production. Figure 1 ).
[0004] Hypophosphite, as a formate analogue, is a competitive inhibitor of formate-metabolizing enzymes (Takamiya 1953). Furthermore, when formate is used as a metabolic intermediate, hypophosphite is an inhibitor of co-cultures of syntrophic butryate oxidizers and hydrogenotrophic methanogens (Sieber, Le, and McInerney 2014). In complex syntrophic communities, formate, hydrogen, and acetate are all important syntrophic products utilized by different types of methanogens. Inhibition of formate metabolism has a feedback effect on the syntrophic community, further inhibiting it, which in turn leads to the accumulation of organic acids, ultimately causing the entire methanogenic system to cease functioning. In other studies, even in complex methanogenic test environments where hydrogenotrophic and methylotrophic methanogens may exist, the inhibition of acetate-utilizing methanogenesis by monofluoroacetate still shows inhibitory effects on methanogenesis (Emptage, Tabinowski, and Odom 1997).
[0005] Previous work has not considered or evaluated the efficacy or specificity of hypophosphite as an inhibitor of methanogenesis. Furthermore, previous studies on the effects of hypophosphite on methanogens examined simple co-cultures of methanogenic archaea and syntrophic bacteria, without considering complex microbial communities, e.g., Sieber, Jessica R., Huynh M. Le, and Michael J. McInerney. 2014. “The Importance of Hydrogen and Formate Transfer for Syntrophic Fatty, Aromatic and Alicyclic Metabolism.” Environmental Microbiology 16 (1): 177–88.
[0006] Relevant literature includes: Pine 1970 The Methane Fermentations, in: Anaerobic Biological Treatment Processes, Chapter 1, p1-10, Advances in Chemistry, Vol.105. Invention Overview
[0008] On the one hand, the present invention provides a method for inhibiting microbial methanogenesis, which includes using a formate analogue—hypophosphite—to inhibit syntrophic methanogenesis in complex environmental microbial communities or the growth of hydrogenotrophic methanogens.
[0009] In the implementation plan:
[0010] The complex environmental microbial communities exist in farmland, wastewater, biological treatment systems such as bioreactors for renewable natural gas (RNG), or natural or managed ecosystems; and / or
[0011] The method also includes obtaining carbon credits by inhibiting methanogenesis.
[0012] On the one hand, the present invention provides a method for modifying hypophosphite-assimilating crop (e.g., rice) plants to improve the efficiency of phosphate fertilizer amendments in farmland while reducing methane emissions. This method includes modifying plant genes to include and express a heterologous hypophosphite / 2-oxoglutarate dioxygenase htxa gene to enable the application of reduced phosphorus, such as hypophosphite, as a phosphorus source.
[0013] In the implementation plan, the crop is rice, corn, wheat, yam, soybean, sugarcane, or cotton.
[0014] On one hand, the present invention provides a method for using reduced phosphorus compounds as phosphate fertilizers for carbon sequestration in deep soils, which includes using reduced phosphorus compounds such as hypophosphite and phosphite fertilizer amendments. These reduced phosphorus compounds are more mobile in soil and sediments but can be used as a phosphorus source for soil microorganisms to increase carbon storage in deep sediments.
[0015] In the implementation scheme, the compound is hypophosphite, the soil is paddy field soil, wherein the hypophosphite applied remains stable in the anaerobic sediment for >6 months.
[0016] This invention encompasses all combinations of the specific embodiments described herein, as each combination has been specifically enumerated. Brief description of the attached diagram
[0018] Figure 1 Carbon and electron flows in an anaerobic methanogenesis system. Arrows indicate fluxes suppressed by inhibitors of formate metabolism, as illustrated in this paper.
[0019] Figure 2 The dose response of hypophosphite to the inhibitory effect of 150 mM formate (gray) or 80% hydrogen (black) on methanococcus marinei S2 cultured as the sole electron donor.
[0020] Figure 3 The dose-response relationship of hypophosphite to the inhibitory effects on the growth and metabolism of nitrate, sulfate, and methanogenic enriched cultures from paddy field sediments using yeast extract as the sole electron donor and carbon source. Hollow symbols represent the overall growth status of the fermentative population in each enrichment, as measured by optical density (OD), while solid symbols represent the production of nitrate, sulfide, or methane, characterizing their respective metabolic activities. The half-maximal inhibitory concentration (IC50) for methane production in methanogenic cultures was 56 μmol, with a 95% confidence interval of 39–78 μmol.
[0021] Figure 4The dose-response relationship of hypophosphite to the inhibitory effects on growth, methanogenic abundance, and methane production in methanogenic enrichment cultures from paddy field sediments using yeast extract as the sole electron donor and carbon source, relative to the untreated control group. Hollow triangles represent the overall growth of fermenting microbiota in each enrichment as measured by optical density (OD), solid triangles represent methane production, and solid squares represent the relative abundance of methanogenic archaea as determined by 16S rDNA amplicon sequencing. The IC50 value for methane production was 56 μmol, with a 95% confidence interval of 39–78 μmol, and the IC50 value for methanogenic abundance was 136 μmol, with a 95% confidence interval of 109–179 μmol.
[0022] Description of specific embodiments of the invention
[0023] Unless otherwise stated or prohibited, in these descriptions and throughout this specification, the terms "a" and "an" mean one or more, and the term "or" means and / or. It should be understood that the embodiments and implementations described herein are for illustrative purposes only and will suggest to those skilled in the art various modifications or alterations thereto, all of which are included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein, including those cited therein, are hereby incorporated in their entirety for all purposes.
[0024] Example 1. Inhibition of microbial methanogenesis by hypophosphite
[0025] Hypophosphite (P is in the +1 oxidation state, H2PO4) 2- ) and phosphite (P is in the +3 oxidation state, HPO) 3- Phosphorus cannot be used as a phosphorus source for plants or animals. Plants or animals only utilize phosphorus in the form of phosphate (P in the +5 oxidation state, PO4). 2- Both phosphites and hypophosphites must be oxidized to phosphates to serve as a phosphorus source and to bind to biomass. The only known pathway for the oxidation of reduced phosphorus is the microbial pathway. Anaerobic phosphite oxidation pathways exist, but for hypophosphites, only aerobic pathways are known. Therefore, phosphites can be a phosphorus source for both aerobic and anaerobic bacteria, but hypophosphites can only be used as a phosphorus source for aerobic bacteria. Hypophosphites are formate analogs and inhibitors of methanogenesis. They are most persistent in anaerobic soils.
[0026] The present invention preferably uses a formate analogue—hypophosphite—to inhibit synergistic methanogenesis. Figure 1We have conducted laboratory tests to demonstrate the selectivity and potency of hypophosphite in different methanogens and methanogenic systems. We disclose, and our results confirm, that hypophosphite has a strong inhibitory effect on hydrogen-trophic methanogens, which produce trace amounts of formate for biosynthetic reactions. Figure 2 Although hypophosphite only inhibits the growth of S2 cultures containing 160 mM formate at concentrations >100 mM, hypophosphite has an inhibitory effect at concentrations below 0.1 mM when S2 is cultured under hydrogen.
[0027] In complex methanogenic communities from paddy soils, we also disclosed and demonstrated the selective inhibitory effect of hypophosphite on methanogenesis relative to fermentation growth, nitrate reduction, or sulfate reduction. Figure 3 We also demonstrated that hypophosphite not only inhibits methanogenesis in the paddy field microbiome, but also inhibits the growth of methanogenic archaea. Figure 4 This selectivity is important for application because hypophosphite has a wide therapeutic window, enabling metabolic shifts to processes other than methanogenesis.
[0028] Practical applications include agriculture, wastewater treatment, bioprocessing, managed ecosystems, and carbon credits. Importantly, hypophosphite is already a safe and effective fertilizer additive and has been approved for use in a variety of agricultural systems, and is present in various environmental systems as it is naturally produced by environmental bacteria (Hanrahan et al. 2005).
[0029] References
[0030] Emptage, Mark, J. Tabinowski, and J. Martin Odom. 1997. “Effect of Fluoroacetates on Methanogenesis in Samples from Selected MethanogenicEnvironments.” Environmental Science & Technology 31 (3): 732–34.
[0031] Hanrahan, Grady, Tina M. Salmassi, Crist S. Khachikian, and KrishnaL. Foster. 2005. “Reduced Inorganic Phosphorus in the Natural Environment:Significance, Speciation and Determination.” Talanta 66 (2): 435–44.
[0032] Liu, He, Jin Wang, Aijie Wang, and Jian Chen. 2011. “ChemicalInhibitors of Methanogenesis and Putative Applications.” Applied Microbiologyand Biotechnology 89 (5): 1333–40.
[0033] Rosentreter, et al. 2021. “Half of Global Methane Emissions Come fromHighly Variable Aquatic Ecosystem Sources.” Nature Geoscience 14 (4): 225–30.
[0034] Sieber, Jessica R., Huynh M. Le, and Michael J. McInerney. 2014. “TheImportance of Hydrogen and Formate Transfer for Syntrophic Fatty, Aromaticand Alicyclic Metabolism.” Environmental Microbiology 16 (1): 177–88.
[0035] Takamiya, Atusi. 1953. “Studies on the Formic Dehydrogenase ofEscherichia Coli III. Determination of the Quantity of the Enzyme within theCell by Using Hypophosphite as a Specific Inhibitor.” Journal of Biochemistry40 (4): 407–14.
[0036] Example 2. Modifying hypophosphite-assimilating rice plants to improve the efficiency of phosphate fertilizer amendments in paddy fields while reducing methane emissions.
[0037] This aspect of the invention provides for the modification of plants to possess the hypophosphite oxidase (htxA) gene, enabling the use of reduced phosphorus compounds as a phosphorus source. The invention is particularly applicable to rice agriculture; see also, for example, the use of hypophosphite as an inhibitor of microbial methanogenesis.
[0038] Phosphites have been used in combination with fertilizers and pesticides in crops that have been engineered to utilize phosphites as a phosphorus source, including rice plants (Lovatt and Mikkelsen 2006; Havlin and Schlegel 2021; Manna et al. 2016). This is achieved by introducing the bacterial ptxD phosphite dehydrogenase gene. Compared to phosphates, phosphites tend to be more mobile in soil (Zhu, Carlson, and Coates 2013) and have a longer half-life. A similar situation exists with hypophosphites; we have also shown that hypophosphites effectively inhibit methanogenesis for several months in batch methanogenic cultures in our laboratory and are essentially unusable as a phosphorus source in an anaerobic gut environment (Rhodehamel, Pierson, and Leifer 1990). Therefore, hypophosphites could provide rice plants with a selective phosphorus source that is not utilized by the plant microbiome while inhibiting methanogenesis.
[0039] This invention describes a method for introducing the hypophosphite / 2-oxoglutarate dioxygenase htxa gene (White and Metcalf 2002) into rice plants using a well-established transformation protocol (Toki et al. 2006). The resulting transgenic rice plants, like their phosphite-utilizing counterparts, are able to utilize hypophosphite as a phosphorus source.
[0040] We have demonstrated that hypophosphite is a selective inhibitor of methanogenesis, readily convertible in rice plants, and that ptxD phosphite dehydrogenase has been shown to function in rice plants. This paper discloses that the introduction of the htxA phosphite dehydrogenase gene confers the ability of rice to utilize hypophosphite. If hypophosphite is used for methane inhibition, this also allows the phosphorus in hypophosphite to be used to support plant growth; that is, by providing hypophosphite as a dual methane inhibitor / phosphorus source, a dual benefit is provided to the rice plant.
[0041] Hypophosphite is cost-effective compared to phosphite, is more stable in anaerobic ecosystems, and can suppress methanogenesis in rice plants. Practical applications include agricultural customers / carbon sequestration.
[0042] References
[0043] Havlin, John L., and Alan J. Schlegel. 2021. “Review of Phosphite as a Plant Nutrient and Fungicide.” Soil Systems 5 (3): 52.
[0044] Lovatt, CJ, and RL Mikkelsen. 2006. “Phosphite Fertilizers: What Are They? Can You Use Them? What Can They Do?” 2006. https: / / spectrumanalytic.com / support / library / pdf / Phosphite_Fertilizers_What arethey.pdf.
[0045] Manna, et al. 2016. “The Development of a Phosphite-MediatedFertilization and Weed Control System for Rice.” Scientific Reports 6(April): 24941.
[0046] Rhodehamel, EJ, MD Pierson, and AM Leifer. 1990. “Hypophosphite: A Review.” Journal of Food Protection 53 (6): 513–18.
[0047] Toki, et al. 2006. “Early Infection of Scutellum Tissue withAgrobacterium Allows High-Speed Transformation of Rice.” The Plant Journal:For Cell and Molecular Biology 47 (6): 969–76.
[0048] White, et al. “Isolation and Biochemical Characterization ofhypophosphite / 2-Oxoglutarate Dioxygenase. A Novel Phosphorus-Oxidizing Enzymefrom Psuedomonas Stutzeri WM88.” The Journal of Biological Chemistry 277(41): 38262–71.
[0049] Example 3. Application of reduced phosphorus compounds as phosphate fertilizer for deep soil carbon sequestration.
[0050] This aspect of the invention provides a method for increasing carbon storage in deep sediments by using hypophosphite and phosphite fertilizer amendments, which are more mobile in soil and sediments but can be used as a phosphorus source for soil microorganisms.
[0051] Increasing the carbon storage capacity of agricultural land could be a nature-based solution to climate change. Stimulating the growth of microorganisms in deep soils to produce stable carbon has been proposed as one approach. Deep carbon generally has a much longer half-life than shallow carbon. Unfortunately, phosphorus and nitrogen are finite in many deep soils and sediments (Jørgensen and Marshall 2016), so most microbial carbon is converted into carbon dioxide and methane through respiration. This limits the accumulation capacity of microbial biomass and increases greenhouse gas emissions from soils and sediments. Microbial respiration metabolism can occur to provide energy for sustaining biomass (Finstad et al. 2023). Fertilizer amendments designed to provide phosphorus and nitrogen to deep soils could circumvent this problem.
[0052] Orthophosphate is the most commonly used fertilizer in agricultural systems. It is bioavailable, but at neutral pH it is a divalent anion and forms tight complexes with divalent cations commonly found in soil, such as Ca, Mg, and Fe. Therefore, it tends to be depleted in deeper sediments. In contrast, phosphites and hypophosphites are monovalent anions and have much higher solubility in the presence of divalent cations. We have demonstrated that phosphites remain dissolved even in high-ferrous sediment columns, thus serving as a phosphorus source for microbial growth (Zhu, Carlson, and Coates 2013). Similar to phosphites, hypophosphites are highly soluble and possess the additional benefit of being a selective inhibitor of methanogenesis, which has a greenhouse effect 10 times greater than that of carbon dioxide. In high-carbon underground environments, methanogenesis is likely to occur when other terminal electron acceptors are depleted.
[0053] Both hypophosphites and phosphites are highly water-soluble and can be applied like any other phosphate fertilizer. Unlike phosphates, they do not bind tightly to soil cations, and therefore migrate into deeper soil layers, such as >6 inches, 12 inches, 18 inches, 24 inches, or 36 inches below the surface. In implementation schemes, hypophosphites can be introduced into paddy fields, ruminant systems, or reactors to suppress methanogenesis. All of these scenarios are anaerobic systems in which hypophosphites can persist for extended periods, such as >6 months.
[0054] Currently, there are no plant methods utilizing hypophosphite (only phosphite-utilizing methods exist). Targeting deep soil and sediment for hypophosphite application is novel. Existing techniques only use surface-applied phosphite for weed control. This invention also differs from plant nutrition or fertilization, and despite the added benefit of suppressing methanogenesis, its objective is to maintain a more soluble form of phosphorus to support deep soil carbon sequestration. Concentration and formulation are adjusted accordingly. Indeed, application to plants can also support microbial biomass; however, the mechanism of anaerobic hypophosphite oxidation is unknown, therefore, in flooded paddy fields, there will not be sufficient oxygen available to support hypophosphite oxidation.
[0055] Our ongoing field validation work in rice, ruminants, and reactor systems supports the efficacy of hypophosphite. For example, we have demonstrated that hypophosphite remains stable in anaerobic sediments for >6 months; see, for example... Figure 2 .
[0056] References
[0057] Finstad, et al. 2023. “Radiocarbon analysis of soil microbial biomassvia direct chloroform extraction.” Radiocarbon, Oct, 1–9.
[0058] Jørgensen, Bo Barker, and Ian P. G. Marshall. 2016. “Slow MicrobialLife in the Seabed.” Annual Review of Marine Science 8: 311–32.
[0059] Zhu, Hongbo, Han K. Carlson, and John D. Coates. 2013. “Applicabilityof Anaerobic Nitrate-Dependent Fe(II) Oxidation to Microbial Enhanced OilRecovery (MEOR).” Environmental Science & Technology 47 (15): 8970–77.
Claims
1. A method for inhibiting microbial methanogenesis, comprising using a formate analogue—phosphotriene—to inhibit the growth of syntrophic methanogenic bacteria or hydrogenotrophic methanogens in complex environmental microbial communities.
2. The method of claim 1, wherein the complex environmental microbial community exists in farmland, wastewater, biological treatment systems, or natural or managed ecosystems.
3. The method of claim 1 or 2, further comprising using the inhibition to obtain carbon credits.
4. A method for modifying hypophosphite-assimilating crop plants to improve the efficiency of phosphate fertilizer amendments in farmland while reducing methane emissions, comprising modifying plant genes to include and express a heterologous hypophosphite / 2-oxoglutarate dioxygenase htxa gene to enable the use of reduced phosphorus, such as hypophosphite, as a phosphorus source.
5. The method of claim 4, wherein the crop is rice, corn, wheat, yam, soybean, sugarcane, or cotton.
6. A method for deep soil carbon sequestration using reduced phosphorus compounds as phosphorus (P) fertilizers, comprising using reduced phosphorus compounds such as hypophosphite and phosphite fertilizer amendments, said reduced phosphorus compounds being more mobile in soil and sediments but serving as a phosphorus source for soil microorganisms to increase carbon storage in deep sediments.
7. The method of claim 6, wherein the compound is hypophosphite, the soil is paddy field soil, and the delivered hypophosphite remains stable in anaerobic sediments for >6 months.