Method for sequestering carbon dioxide

Abstract

A method for sequestering carbon dioxide (CO2) includes forming a mixture by combining a sulfate salt source with an organic matter source under anaerobic conditions, reacting at least one trained microbial community with the mixture in a manner that causes the sulfate salt source and organic matter source to be converted into hydrogen sulfide, sulfate salt cations, and bicarbonate ions, and sequestering CO2 as an insoluble carbonate salt by reacting the sulfate salt cations with the bicarbonate ions that forms the insoluble carbonate salt, CO2, and water. A method for producing trained microbial communities includes creating a seed by collecting at least one microbial community, culturing a microbial community collected to produce a seed community, training the seed community for converting sulfate salts and organic matter to alkaline and sulfidic solutions to produce a trained seed community, and selecting a trained microbial community from the trained seed community.

Description

0050.2404001 (MIT-26176)METHOD FOR SEQUESTERING CARBON DIOXIDERELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 703,077, filed on October 3, 2024. The entire teachings of the above application(s) are incorporated herein by reference.BACKGROUND

[0002] To meet global phosphorus demands for fertilizer production, phosphate rock (apatite) is treated with sulfuric acid to produce phosphoric acid. This process generates phosphogypsum, a waste product primarily composed of gypsum (CaSO4 ■ 2H2O) with trace impurities, such as fluoride and radioactive elements like uranium and thorium.Phosphogypsum presents significant environmental challenges: approximately 300 million tons are produced annually, and 86% is either stacked indefinitely or discharged into the ocean1. Additionally, there is waste gypsum produced from flue gas desulfurization and titanium dioxide production2,3. The associated disposal issues have motivated the search for economically viable strategies to valorize waste gypsum.

[0003] There is a need for reducing produced waste gypsum utilizing reactors and methods.SUMMARY

[0004] Disclosed herein is a method for sequestering carbon dioxide (CO2) by forming a mixture by combining a sulfate salt source with an organic matter source under anaerobic conditions, reacting at least one trained microbial community with the mixture in a manner that causes the sulfate salt source and organic matter source to be converted into hydrogen sulfide (H2S), sulfate salt cations, and bicarbonate ions (HCO3 ), and sequestering carbon dioxide as an insoluble carbonate salt by reacting the sulfate salt cations with the bicarbonate ions that forms the insoluble carbonate salt, carbon dioxide, and water.

[0005] In some instances, the at least one trained microbial community reduces sulfate ions in the absence of methanogens. In some instances, the organic matter source may be at least one of organic waste, sewage, animal manure, compost waste, agricultural waste, food14229050. vl0050.2404001 (MIT-26176) processing residues, papermill sludge, brewery waste, meat processing waste, wastewater, natural organic material, leaf litter, wood, corn stalks, or any combination thereof. In some instances, the sulfate salt sources may be industrial waste gypsum, phosphogypsum, flue gas desulfurization (FGD) gypsum, natural gypsum, titanium gypsum, anhydrite, barite, epsomite, celestinejarosite, calcium sulfate sources or anhydrides thereof, barium sulfate sources or anhydrides thereof, magnesium sulfate sources or anhydrides thereof, copper (II) sulfate sources or anhydrides thereof, lead (II) sulfate sources or anhydrides thereof, strontium sulfate sources or anhydrides thereof, or any combination thereof.

[0006] Also disclosed herein is a method for producing trained microbial communities by creating a seed by collecting at least one microbial community, culturing the at least one microbial community collected to produce a seed community, training the seed community for converting a sulfate salt source and organic matter source to alkaline solutions and sulfidic solutions to produce a trained seed community, and selecting a trained microbial community from the trained seed community. In some instances, the trained microbial community reduces sulfate ions without generating methane. In some instances, the at least one microbial community is enriched by applying a selective pressure.

[0007] Further disclosed herein is a bioreactor that has a multi-phase inlet for a sulfate salt source and an organic matter source, at least one trained microbial community, and an anaerobic chamber within the bioreactor.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0009] FIG. l is a general diagram of a reactor with multi-phase inlets for sequestering carbon from organic matter.

[0010] FIGs. 2A-2C are graphs showing evolution of solution chemistry in trained and untrained communities. A) average sulfide concentrations, B) average methane concentrations, C) average alkalinity concentrations (ppm as CaCO3). Red circles represent Trained Community #1, green triangles represent Trained Community #2, and blue squares represent the untrained community. Averages shown are of triplicate serum bottles, the error24229050. vl0050.2404001 (MIT-26176) bars are standard deviation, and small low opacity shapes represent raw values of each replicate.

[0011] FIG. 3 is a graph comparing the average methane and sulfide concentrations in diluted and undiluted concentrated sewage sludge. Red circles represent Trained Community #1, green triangles represent Trained Community #2, and blue squares represent the untrained community. Gray dashed arrows represent the COD change from ~45 g / L to ~1.5 g / L. Data points show average values from triplicate culture bottles on day 8 of incubation.

[0012] FIGs. 4A-4C are graphs of A) Average calcium to total sulfur ratio (Ca: Total S) normalized to expected equilibrium (see Exemplification) and concentrations of B) average calcium and C) average sulfate over time. Red circles represent Trained Community #1, green triangles represent Trained Community #2, and blue squares represent the untrained community. The dashed black line in FIG. 4A represents a ratio of one, where values below the line represent an observed calcium deficit from the expected ratio after assumed equilibrium by day six. Averages shown are of triplicate serum bottles, the error bars are standard deviation, and small low opacity shapes represent raw values of each replicate.

[0013] FIG. 5 is a graph of bacterial orders represented by the 16S rRNA sequences in triplicate cultures of trained and untrained communities on day seventeen.

[0014] FIGs. 6A and 6B are graphs of microbial families represented by the 16S rRNA sequences in averaged triplicate cultures of trained and untrained communities on day seventeen. A) Sulfate-reducing bacteria, B) methanogenic archaea.

[0015] FIG. 7 is a schematic of the inferred microbial interactions that govern the partitioning of electrons. Low organic loading = 1.5 g / L, high organic loading = 45 g / L. TC#1 = inoculated with trained community #1, TC#2= inoculated with trained community #2, UT = untrained, no inoculum added, it is rather just a native sewage community encountering sulfate for the first time. Ferm. = fermenter, MPA = methane producing archaea, SRB = sulfate reducing bacteria, VFA = volatile fatty acids. The benzene ring represents general aromatics including other forms. Acetate is included separately from other VFAs in the top left section to emphasize the reliance on acetate for methanogens in noncompetitive substrate use conditions.

[0016] FIGs. 8A and 8B are graphs showing the concentration of sulfide (FIG. 8A) and concentration of alkalinity (FIG. 8B) of an untrained microbial community and two different trained microbial communities over time.34229050. vl0050.2404001 (MIT-26176)DETAILED DESCRIPTION

[0017] A description of example embodiments follows.

[0018] As used herein, the term “about” means within an acceptable error range for the particular value, as determined by one of ordinary skill in the art. Typically, an acceptable error range for a particular value depends, at least in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of ± 20%, e.g., ± 10%, ± 5% or ± 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Exemplification section hereinbelow.

[0019] Proposed solutions of reducing produced waste gypsum — such as incorporating waste gypsum into road materials or using it as a soil amendment — face environmental and scalability challenges4. An alternative approach is gypsum bioconversion that involves microbial sulfate reduction (MSR), in which sulfate-reducing bacteria convert sulfate from gypsum to sulfide, releasing calcium and alkalinity into solution5. MSR relies on using molecular hydrogen or simple organic molecules such as acetate, as shown in the reaction:

[0020] The resulting calcium- and sulfide-rich solution can then be used in downstream processes: sulfide can be converted to elemental sulfur (via abiotic or biological oxidation610), and calcium and alkalinity can precipitate as carbonate minerals, offering a route for carbon sequestration.

[0021] A major challenge in implementing this process is the availability of low-cost electron donors for MSR5,9,11. Municipal sewage presents a promising solution5. In modem wastewater treatment, sewage sludge is typically digested anaerobically, where organic matter is converted into methane via methanogenesis. This methane is often vented or flared to CO2, contributing to greenhouse gas emissions12.

[0022] Integrating waste gypsum bioconversion and sulfate reduction into existing sewage treatment infrastructure offers a pathway to mitigate these emissions13 l 8. By adding gypsum directly to anaerobic digesters, electron donors could be redirected from methane production to sulfate reduction. The present disclosure addresses the initial step in developing such an integrated system, aiming to convert gypsum using municipal sewage as both a carbon source and the source of microbial diversity for sulfate reducing bacteria. The goal is44229050. vl0050.2404001 (MIT-26176) to generate alkaline solutions for downstream carbon sequestration and elemental sulfur recovery.

[0023] Extensive research has focused on optimizing microbial communities for anaerobic digestion of sewage sludge through methanogenesis due to its widespread use in wastewater treatment plants; key operational controls include inoculum -to-substrate ratios, upward fluid velocity to retain biomass, and substrate recirculation ratios19 25. However, there remains a need for studies of anaerobic digestion of sewage sludge through MSR, especially those that aim to minimize methane emissions while enhancing sulfate reduction and alkalinity generation.

[0024] Prior work demonstrated bioconversion of waste gypsum using sewage sludge, but did not characterize the microbial community that mediated this process5. Other research has optimized carbon loading conditions to influence the balance between methanogenesis and sulfate reduction26 30, though largely without characterizing the inoculum or community structure. A recent study has quantified bioconversion kinetics in an optimized reactor using nutrient-rich medium instead of sewage sludge9. Other efforts have characterized microbial communities and inoculum source effects in synthetic nutrient-rich systems31,32, but none have trained communities on sewage sludge while measuring methane emissions. In general, inoculum compositions and enrichment processes are either unreported or not connected to functional outcomes. Most research to date also does not measure methane production during gypsum treatment5 9 1 1 31 41or reports it without attempting to suppress or relate it systematically to the rates of sulfate reduction, alkalinity generation, or calcium concentrations in the solution42 45.

[0025] The rates of organic compound oxidation and subsequent methane and sulfide production are driven by the abundance of organic substrates and relative abundance of sulfate, including complex plant biomass and labile compounds like acetate, collectively referred to as organic loading. This loading is a well-established control on anaerobic redox processes: sulfate reducing bacteria typically outcompete methanogens for electron donors under conditions of low organic loading46, where input is often very limited between near 0 and 1.5 g COD / L. In contrast, environments with high organic loading — such as eutrophic sediments or sulfate-rich sewage sludge with up to 90 g COD / L — can support both metabolisms simultaneously47 50. However, how electron donors are partitioned between these competing pathways in systems with high organic loading remains unclear.54229050. vl0050.2404001 (MIT-26176)

[0026] The aim was to enrich and optimize a microbial community that rapidly reduces sulfate using organic electron donors from municipal wastewater, generates alkalinity, and minimizes methane production. A community was identified by comparing the evolution of solution chemistry in systems inoculated with two distinct microbial enrichments to that in systems inoculated with the indigenous microbial population from sewage sludge, under both high and low organic loading. Analyses of microbial diversity indicate that interactions among specific taxa may govern the partitioning of electron donors between sulfate reduction and methanogenesis. Differences in this partitioning across communities highlight the importance of using trained inocula when seeking to control competition and scale inherently unsterile systems. These insights inform the design of a gypsum bioconversion process that mitigates methane emissions by favoring sulfate reduction and maximizing alkalinity generation.Methods of Sequestering Carbon Dioxide

[0027] Described herein is a method for sequestering carbon dioxide (CO2). The method may include (a) forming a mixture by combining a sulfate salt source with an organic matter source under anaerobic conditions, (b) reacting at least one trained microbial community with the mixture in a manner that causes the sulfate salt source and organic matter source to be converted into hydrogen sulfide (H2S), sulfate salt cations, and bicarbonate ions (HCO3 ), and (c) sequestering carbon dioxide as an insoluble carbonate salt by reacting the sulfate salt cations with the bicarbonate ions that forms the insoluble carbonate salt, carbon dioxide (CO2), and water. In some aspects, the method may include concentrating the mixture and repeating (b) and (c).

[0028] In some aspects, the organic matter source may be organic waste, sewage, animal manure, compost waste, agricultural waste, food processing residues, papermill sludge, brewery waste, meat processing waste, wastewater, natural organic material, leaf litter, wood, corn stalks, or any combination thereof. In some further aspects, the organic matter source described herein may undergo a pre-step or pretreatment before being incorporated into the method described herein. In some further aspects, the pre-step or pretreatment may include at least a chemical reaction, a mechanical modification (i.e. grinding, dissolving, diluting, concentrating, heating, cooling, drying, separating, combining), biologically degrading, filtering, or a combination thereof.64229050. vl0050.2404001 (MIT-26176)

[0029] In some aspects, the sulfate salt source may be industrial waste gypsum, phosphogypsum, flue gas desulfurization (FGD) gypsum, natural gypsum, titanium gypsum, anhydrite, barite, epsomite, celestinejarosite, calcium sulfate sources or anhydrides thereof, barium sulfate sources or anhydrides thereof, magnesium sulfate sources or anhydrides thereof, copper (II) sulfate sources or anhydrides thereof, lead (II) sulfate sources or anhydrides thereof, strontium sulfate sources or anhydrides thereof, or any combination thereof. In some further aspects, the sulfate salt source may be industrial waste gypsum, phosphogypsum, flue gas desulfurization (FGD) gypsum, natural gypsum, or a combination thereof.

[0030] In some aspects, the at least one trained microbial community reduces sulfate ions without generating methane. The term “without generating methane” refers to producing about 0% yield methane to about 2% yield methane relative to the total products produced. In some other aspects, the at least one trained microbial community does not produce methane when the concentration ratio of organic matter source to sulfate salt source is about 0.5 to about 5. In some further aspects, the concentration ratio of organic matter source to sulfate salt source may be about 0.8 to about 4. In some further aspects, the concentration ratio of organic matter source to sulfate salt source may be from about 0.9 to about 3.7. In some further aspects, the concentration ratio of organic matter source to sulfate salt source may be from about 0.9 to about 3.7 and the concentration of organic matter source may be from about 0.8 g / L to about 3.7 g / L. In some aspects, the at least one trained microbial community does not produce methane when the ratio of chemical oxygen demand to sulfate is from about 0.9 to about 3.7 and the organic matter has a concentration from about 0.8 g / L to about 3.7 g / L. In some aspects, the at least one trained microbial community may have sulfate reducing bacteria, fermenting bacteria that produce substrates for sulfate reducing bacteria and a reduced contribution of hydrogenotrophic methanogenic archaea relative to the untrained communities. In some further aspects, the sulfate reducing bacteria may have more Desulfomicrobiaceae, Desulfurisporaeceae, Desulfobacterales, or a combination thereof, and fewer Methanobacteriaceae and Methanomassiliaceae than untrained microbial communities. In some other aspects, the at least one trained microbial community may be enriched by applying selective pressure. In some further aspects, the selective pressure that enriches the at least one trained microbial community may include the addition of sulfate or sulfate-bearing minerals, the addition of specific organic matter sources, elevate74229050. vl0050.2404001 (MIT-26176) temperatures, atmospheric exposure, addition of sulfide to the microbial community, or any combination thereof.

[0031] Sulfate reducing bacteria” perform microbial sulfate reduction, which is a metabolic pathway of the bacteria. The bacteria in the microbial communities consume an organic substrate (i.e. an organic matter source) and sulfate ions (SO42) under anaerobic conditions to produce hydrogen sulfide (H2S), carbon dioxide, and water. This is an anaerobic respiration, as the bacteria consume the sulfate ions instead of oxygen, as the sulfate ions act as the terminal electron acceptor in the process.

[0032] The microbial sulfate reduction performed by the trained microbial communities may use organic electron donors from the organic matter source and the sulfate ions to produce bicarbonate (HCO3 ) ions and may release the cation from the sulfate salt source into the solution. Introducing these two ions into solution then forms an insoluble carbonate salt, carbon dioxide, and water. An example embodiment may be with calcium ions, which would have the following chemical reaction: Ca2++ 2 HCCh —> CaCCh + CO2 + H2O. This reaction forms an insoluble carbonate salt that prevents a second molar equivalent of carbon dioxide from being released into the atmosphere due to its stability in the presence of water, effectively cutting the carbon dioxide produced from the waste in half.

[0033] In some aspects, the method may further include separating the carbonate salt and hydrogen sulfide. In some further aspects, the method may further include collecting the carbonate salt and repurposing same for industrial applications. In some aspects, the industrial applications may include adding the carbonate salt to construction materials, soil conditioning, or adding the carbonate salt to seawater or other bodies of water to increase alkalinity.

[0034] In some aspects, the method may further include oxidizing the hydrogen sulfide in a manner that forms elemental sulfur (S°). In some further aspects, the oxidizing may be performed via precipitation as metal sulfides. In some further aspects, the metals used for precipitating may include, but are not limited to: copper, zinc, cadmium, lead, nickel, cobalt, mercury, silver, arsenic, antimony, sodium, ammonium, iron, calcium, barium, or a combination thereof. In some other aspects, the oxidizing may be performed via microbiological metabolism. In some instances, the microbiological metabolism may be performed using sulfur-oxidizing bacteria. In some other instances, the oxidizing is performed via a biological or abiotic system.84229050. vl0050.2404001 (MIT-26176)

[0035] Hydrogen sulfide that is produced from the method herein may also be managed using gas-stripping. Gas-stripping removes the hydrogen sulfide from a container or reactor using a biological or abiotic process. Metal precipitation, as described herein, converts the hydrogen sulfide into metal sulfides for preventing sulfide toxicity.

[0036] In some aspects, the method may be performed in a bioreactor. In some further aspects, the bioreactor may be an upflow anaerobic sludge blanket (UASB) reactor, fluidized bed reactor, packed bed reactor, membrane bioreactor, sequencing batch reactor, or anaerobic digester. In some other instances, the method may be performed underground.

[0037] An upflow anaerobic sludge blanket (UASB) reactor operates by intaking the sulfate salt source and organic matter source and flowing the sulfate salt source and organic matter source upward through a sludge blanket containing the trained microbial communities that reduce sulfate to hydrogen sulfide an insoluble carbonate salt forms in the reactor. A “sludge blanket” refers to a suspended bed of active microbial biomass that accumulates at the bottom and middle of the reactor that are in granular particles of compact microbial aggregates. This reactor is beneficial due to its high sulfate reduction efficiency, biogas production, and ease of sludge management.

[0038] A fluidized bed reactor operates by introducing the sulfate salt source and organic matter source in a combined solution into the reactor containing the trained microbial communities in suspended particles. This precipitates the insoluble carbonate salt from the media within the reactor. This reactor is beneficial due to its high biomass density, enhanced mass transfer, and is resistant to clogging.

[0039] A packed bed reactor operates by flowing a sulfate salt source and organic matter source in a combined solution into a reactor containing packed media bed with biofilms of the trained microbial communities. The insoluble carbonate salt formed precipitates within the packed media bed. This reactor is beneficial due to the large surface area for microbial biofilm growth, effectively handling sulfate salt source, and solid-liquid separation.

[0040] A membrane bioreactor operates by filtering a solution of sulfate salt source and organic matter source through a membrane with the trained microbial communities that separates the liquid and formed insoluble carbonate salt. This reactor is beneficial for high quality effluent, small footprint, and minimal sludge loss.

[0041] A sequencing batch reactor operates by treating a solution of sulfate salt source and organic matter source with the trained microbial communities in batch cycles. This reduces the sulfate salt source to an insoluble carbonate salt, which precipitates and is94229050. vl0050.2404001 (MIT-26176) decanted each batch cycle. This reactor is beneficial due to flexible operation and efficient handling of variable solid and liquid loads.

[0042] FIG.1 shows a schematic of the overall method for sequestering carbon dioxide. The organic matter source 110 is added to the container 113 under anaerobic conditions that is containing the at least one trained microbial community 112. The sulfate salt source 112 is also added to the container 113. Once both the organic matter source 110 and sulfate salt source 112 are present, the at least one trained microbial community 112 metabolizes both sources and undergoes sulfate reduction, which forms an insoluble carbonate salt 114a, carbon dioxide 114b, water, and hydrogen sulfide. The carbon dioxide formed is half on a molar ratio compared to untrained microbial communities. The hydrogen sulfide and insoluble carbonate salt 114a are separated and removed from the container 113, where the hydrogen sulfide is oxidized into elemental sulfur 115 and the insoluble carbonate salt 114a is used for industrial applications which may include construction materials 116a or agricultural fertilizers 116b.

[0043] Also described herein is an alternative method for sequestering carbon dioxide (CO2). The method may include (a) forming a mixture by combining a sulfate salt source with an organic matter source under anaerobic conditions and (b) reacting at least one trained microbial community with the mixture in a manner that causes the sulfate salt source and organic matter source to be converted into hydrogen sulfide (H2S), sulfate salt cations, and bicarbonate ions (HCO3 ) while maintaining a pH that solubilizes bicarbonate ions. The solution may then be disposed of into a larger body of water that maintains a pH that solubilizes bicarbonate ions. A pH that solubilizes bicarbonate ions ranges from about 6.5 to about 8.3. As the pH approaches neutral to acidic, the bicarbonate ions will gradually principate with cations that form an insoluble carbonate salt.

[0044] Aspects of this section described herein are also applicable throughout the present disclosure, including sections “Bioreactor for Sequestering Carbon Dioxide” and “Methods for Producing Trained Microbial Communities.”Bioreactor for Sequestering Carbon Dioxide

[0045] Described herein is a bioreactor for sequestering carbon dioxide. The bioreactor may have a multi-phase inlet to the bioreactor, at least one trained microbial community, and an anaerobic chamber within the bioreactor. The multi-phase inlet may have a sulfate salt source and an organic matter source.104229050. vl0050.2404001 (MIT-26176)

[0046] A “multi-phase inlet” describes an inlet capable of handling solids, liquids, and / or gases through the inlet into a container that may pump the solids, liquids and / or gases through the inlet and into a container, which may be a bioreactor. In some instances, solids may include an organic matter source, a sulfate salt source, or a combination thereof. In some instances, liquids may include an organic matter source, a sulfate salt source, or a combination thereof. In some instances, gases may include inert gases like nitrogen or argon gas. In some instances, the multi-phase inlet may introduce natural gypsum, industrial waste gypsum, or a combination thereof, with city sewage, animal manure, or a combination thereof into the bioreactor, thereby introducing a sulfate salt source and an organic matter source into the bioreactor.

[0047] In some aspects, the bioreactor may also have at least one container separating a carbonate salt, hydrogen sulfide, or a carbonate salt and hydrogen sulfide.

[0048] In some aspects, the bioreactor may be an upflow anaerobic sludge blanket reactor, fluidized bed reactor, packed bed reactor, membrane bioreactor, sequencing batch reactor, or anaerobic digester.

[0049] Aspects that overlap with the “Methods for Sequestering Carbon Dioxide” are applicable to this section. The aspects described herein include, but are not limited to, organic matter sources and sulfate salt sources.Methods for Producing Trained Microbial Communities

[0050] Described herein is a method for producing trained microbial communities. The method may include creating a seed by collecting at least one microbial community, culturing the at least one microbial community collected to produce a seed community that is trained for converting a sulfate salt source and organic matter source to alkaline solutions and sulfidic solutions to produce a trained seed community, and selecting a trained microbial community from the trained seed community.

[0051] An “alkaline solution” refers to a liquid that has pH of greater than 7, sometimes also referred to as a “basic solution.” The pH is greater than 7 due to the hydroxide (OH ) ion concentration being greater than H+concentration. In some instances, the liquid may be water, an organic solvent, or a miscible combination thereof. In some instances, the pH is greater than 7 due to the formation of bicarbonate (HCO3 ) ions, which are in part formed from taking a hydrogen from water, which increases the hydroxide ion concentration.114229050. vl0050.2404001 (MIT-26176)

[0052] A “sulfidic solution” refers to a liquid that has the presence of sulfide (S2‘) ions present within the liquid, either dissolved or suspended. In some instances, the liquid may be water, an organic solvent, or a miscible combination thereof. In some further instances when the liquid has water, the sulfidic solution may have dissolved hydrogen sulfide (H2S).

[0053] In some aspects, the trained microbial community may reduce sulfate ions without generating methane. The term “without generating methane” refers to producing about 0% yield methane to about 2% yield methane relative to the total products produced. In some other aspects, the at least one trained microbial community does not produce methane when the concentration ratio of organic matter source to sulfate salt source is about 0.5 to about 5. In some further aspects, the concentration ratio of organic matter source to sulfate salt source may be about 0.8 to about 4. In some further aspects, the concentration ratio of organic matter source to sulfate salt source may be from about 0.9 to about 3.7. In some further aspects, the concentration ratio of organic matter source to sulfate salt source may be from about 0.9 to about 3.7 and the concentration of organic matter source may be from about 0.8 g / L to about 3.7 g / L. In some aspects, the at least one trained microbial community does not produce methane when the ratio of chemical oxygen demand to sulfate is from about 0.9 to about 3.7 and the organic matter has a concentration from about 0.8 g / L to about 3.7 g / L. In some aspects, the at least one trained microbial community may have sulfate reducing bacteria, fermenting bacteria that produce substrates for sulfate reducing bacteria and a reduced contribution of hydrogenotrophic methanogenic archaea relative to the untrained communities. In some further aspects, the sulfate reducing bacteria may have more Desulfomicrobiaceae, Desulfurisporaeceae, Desulfobacterales, or a combination thereof, and fewer Methanobacteriaceae and Methanomassiliaceae than untrained microbial communities. In some other aspects, the at least one trained microbial community may be enriched by applying a selective pressure. In some further aspects, the selective pressure that enriches the at least one trained microbial community may include the addition of sulfate or sulfate-bearing minerals, the addition of specific organic matter sources, elevate temperatures, atmospheric exposure, addition of sulfide to the microbial community, or any combination thereof.

[0054] In some aspects, the collecting of at least one microbial community is from rocks, minerals, organic material, water, or a combination thereof. In some further aspects, the collecting of at least one microbial community is from sediments. The term “sediments” refers to particles of rock, minerals, organic material, or a combination thereof that have been124229050. vl0050.2404001 (MIT-26176) exposed to weathering processes (i.e. wind, water, ice, erosion). Sediments can typically be found in bodies of water or near bodies of water and vary in size, shape, and texture. In some further aspect, at least one microbial community is collected from organic material, natural environments, or a combination thereof.EXEMPLIFICATION

[0055] Microbial conversion of industrial waste gypsum — such as the 300 million tons of hazardous phosphogypsum stacked or discharged annually — using municipal sewage sludge potentially can sequester carbon by replacing methanogenesis with sulfate reduction in sludge digestion. Although the availability of organic carbon influences terminal metabolism, it remains unclear whether methane emissions during gypsum treatment are primarily driven by community composition or organic availability. By comparing metabolic fluxes toward sulfate or methane in trained and untrained communities grown on different organic loads, this disclosure shows that community composition, biomass, and organic availability collectively determine sulfide and methane flux by shaping fermentative structure and syntrophic competition. One of the trained communities reduced more sulfate (47 mg SO42' / L / day) and produced much less methane (less than 1 mg CEL / L / day) than native sewage sludge communities, accumulating 41 mg alkalinity as CaCOs / L / day. Lower organic loads increased the importance of syntrophy and competition between sulfate reducing bacteria and methanogens due to scarcity of substrates. The untrained community exhibited lower sulfate reduction rates and moderate methane production compared to the trained communities, highlighting the importance of selective enrichment in efficient bioconversion of waste gypsum. The production of different amounts of methane at similar sulfate reduction rates in two different trained communities revealed the importance of microbial ecology. These composition of the two communities suggested that sulfate reducing bacteria in the community that generated less methane outcompeted methanogens for acetate and EE, while sulfate reducers and methanogens coexisted because of the non-competitive substrate use in the cultures that generated more methane. Targeted enrichments and controlling the organic loading may improve efforts to suppress methane production and efficiently convert gypsum to sulfide and alkaline-rich solution in sludge treatment systems.Methods134229050. vl0050.2404001 (MIT-26176)

[0056] Experimental setup: Thickened sewage sludge and final treated effluent (FEFF) were sourced from Deer Island Wastewater Treatment Plant, operated by the Massachusetts Water Resources Authority (MWRA). Thickened sewage sludge had a chemical oxygen demand (COD) ranging from 74 to 92 g / L. A concentrated sewage medium, referred to as “high organic loading” conditions, was prepared by a 1 : 1 dilution of sewage sludge with FEFF and sieved through a 0.35 mm mesh. A dilute sewage medium, referred to as “low organic loading” conditions, was prepared by another 30.7x dilution of the concentrated sewage medium, resulting in a final 61.4x dilution. The media with high and low organic loading were amended by 22.6 g / L and 5.18 g / L of pure gypsum, respectively. These concentrations are equivalent to a total sulfate concentration of 131.3 mM and 30.1 mM, respectively. All incubations were conducted in triplicate with 10% v / v inoculum and incubated at 32°C, shaking at 135 rotations per minute (rpm) in the dark. The inocula contained enriched communities that had been grown for two weeks on the sewage media that matched the media used in that experiment. While initial biomass between treatments was variable, the degree of sulfide production was allowed to progress to similar stages before inoculating for experiments; after inoculation of trained communities, initial sulfide concentrations ranged between 0.16-0.29 mM for high organic loading experiments and 0.47-0.75 mM for low organic loading experiments. The high organic loading experiment is reported up to 8 days of incubation, and the low organic loading experiment is 17 days of incubation.

[0057] Enrichments: This disclosure utilized two trained communities and an untrained community which consisted of microbes that were present in the untreated sewage sludge and had no prior exposure to sulfate-rich conditions. The main environmental difference between the trained communities is the organic loading at which they were enriched.

[0058] The enrichment process for Trained Community #1 sampled sulfide-rich sediment from Plum Island, MA, USA, and added it to sewage medium of 2500 mg / L COD amended with 20 mM sodium sulfate. Sulfide and sulfate concentrations were monitored during each generation of enrichment, and cultures were transferred to new medium (10% inoculation) when sulfide and sulfate concentrations began to plateau. The Trained Community #1 used in this manuscript was harvested after 3 times of transfer of the initial enrichment culture.

[0059] The enrichment process for Trained Community #2 used inoculum from a sulfide- rich stream from a sulfur mine in Sicily, Italy, which was added to ~260x diluted and filtered sewage with flue-gas desulfurization gypsum. Sulfide concentrations were monitored during144229050. vl0050.2404001 (MIT-26176) the first generations of enrichment, and a new generation was inoculated on ~260x diluted and 0.8 gm filtered sewage once sulfide concentrations begin to plateau. This enrichment process was repeated ten times and as experiments continued.

[0060] Sulfide concentrations: Sulfide concentrations were measured by the Cline assay51. Generally, samples were diluted lOOx in 0.05M zinc acetate and measured immediately.

[0061] Methane concentrations: Methane concentrations were measured using a Shimadzu GC-2014 gas chromatograph with a PDHID and helium carrier gas. The pressure in the culture vials with high organic loading was relieved by degassing. Therefore, methane in those cultures could only be measured as the difference of methane concentration in the relieved sample and the methane in the pressurized sample after incubation. It was presumed that any increase in pressure during that time frame is negligible and thus assume 1 atm. Dissolved methane [CFUaq], in mM, was calculated as:

[0063] where PCH4 is the partial pressure (ppm) of methane assuming 1 atm total pressure, Vh is the headspace volume, R is the universal gas constant (L atm / mol K), T is the temperature (K), Vs is the volume of the medium containing sewage and gypsum, and 1000 is the conversion factor for mM.

[0064] Alkalinity and pH: Alkalinity was measured using a ThermoScientific Orion Total Alkalinity Test Kit (Cat. No. 700010TS). The pH was measured using Vernier Triscompatible flat pH sensors or AtlasScientific EZO pH sensors.

[0065] Ion concentrations: Sulfate, calcium, and magnesium were measured using a dual-channel Shimadzu ion chromatograph. Cations were measured with the Shodex “Monovalent and Divalent Cation Standards (4) (YK-421) (Phosphoric Acid Eluent)” method, and anions with the Shodex “Anion Standards (19) Rapid Analysis (SI-90 4E)” method, with the column temperature set to 40°C.

[0066] Electron partitioning towards sulfate reduction and methanogenesis: To monitor how energy as electron donors is partitioned towards sulfate reduction, this disclosure calculated a molar ratio of sulfide produced over the sum of sulfide produced and methane produced. Thus, as electron partitioning was calculated assuming energy usage was divided between sulfate reduction and methanogenesis, this disclosure calculated the partitioning of electron donors towards methanogenesis as 1 minus the previously calculated ratio towards sulfate reduction. Electron partitioning was calculated for high-organic loading154229050. vl0050.2404001 (MIT-26176) conditions from incubation days 3 - 8 (n = 2 - 3 replicates). Relative percent increase in electron partitioning towards sulfate reduction compared to the untrained community was calculated by taking the quotient of the respective fractions of electron donors towards sulfate reduction.

[0067] Total and volatile solids (TS and VS): TS and VS were measured using Standard Methods 2540 SOLIDS (23rded, 2017). Briefly, final culture sludge was dried at 105°C and percent total solids were calculated as the dry mass of the sample normalized to the total wet mass of the sample. VS powder was prepared by anaerobically drying the entire incubation slurry at the end of the experiment. VS was combusted at 550°C and the resulting mass loss was normalized to the dry mass. Liquid was not separated from solids before drying, and the dried powder was reused for X-ray diffraction (XRD) analysis. “% VS removal” is calculated as (mass of initial VS - mass of final VS) / mass of initial VS.

[0068] Calcium to Total S ratio (Ca: Total S): The ratio of calcium to total sulfur was calculated by dividing measured calcium concentrations by the sum of sulfate and sulfide concentrations. To track potential carbonate precipitation, the Ca: Total S ratio at day 6 as a reference point and scaled it by changes in total sulfur to calculate an expected ratio over time was used. Measured Ca: Total S values were then normalized to this expectation, with values below 1 interpreted as indicative of calcium removal, potentially due to carbonate precipitation. Calcium carbonate is the thermodynamically favored precipitate under conditions described herein, and no magnesium carbonates were detected by XRD, so Ca: Total S is used here to infer the onset and extent of carbonate mineral formation (FIGs. 4A- 4C). Ratios were only calculated from day 6 onward due to non-stoichiometric Ca: SO- ' ratios observed earlier in the incubation.

[0069] 16S rRNA gene assays: DNA was extracted using the DNeasy PowerSoil Pro Kit(Qiagen, Hilden, Germany) and quantified with Qubit dsDNA HS Assay Kit and Qubit 3 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed on an Illumina MiSeq sequencer using universal primers U515F (5'- GTGCCAGCMGCCGCGGTAA-3') and E786R (5'-GGACTACHVGGGTWTCTAAT-3'), targeting the V4 region. Sequences were processed with the DADA2 pipeline, and taxonomy was assigned using the MiDAS reference database version 5.3. Raw sequence data are available at ENA project accession PRJEB89716, which is known in the art.Results164229050. vl0050.2404001 (MIT-26176)

[0070] It was hypothesized that concentrated sewage sludge with added gypsum would support both sulfate reduction and methanogenesis, but that the partitioning of electron donors between these competing metabolisms would differ depending on inoculum history. It was further hypothesized that the addition of an external inoculum would be less impactful for determining competition between sulfate reduction and methanogenesis in experiments with high organic loading because abundant organic substrates would reduce the importance of microbial competition. To test these hypotheses, triplicate cultures of the two trained communities and an untrained community in concentrated sewage sludge (~45 g COD / L) amended with excess gypsum were incubated. The trained communities had previously been enriched by serial passaging on sewage in the presence of sulfate. This process favors sulfatereducing bacteria (SRB) over methanogens by shaping fermentative substrate availability early during enrichment. The untrained community consisted of microbes that were present in the untreated sewage sludge and had no prior exposure to sulfate-rich conditions.

[0071] All treatments exhibited simultaneous sulfate reduction and methane production in the presence of high organic loading. Both trained communities diverted more electron donors toward sulfate reduction than the untrained community (Table 1), suggesting that enrichment history can shift microbial function to favor gypsum conversion in the presence of high organic loading. Trained communities also generated 3-15% more alkalinity than the untrained community (FIG. 8B); however, persistent methane production limited sulfate reduction efficiency by competing for available electron donors. These tradeoffs motivated a second experiment under conditions designed to suppress methanogenesis and amplify the importance of inoculum-driven community structure.

[0072] Table 1 : Partitioning of competing metabolisms under high organic loading conditions.

[0073] To overcome the persistent methane production in the presence of high organic loading and shift electron donors toward sulfate reduction, the organic loading was lowered. It was hypothesized that as decreased organic substrate availability will suppress174229050. vl0050.2404001 (MIT-26176) methanogenesis and enhance sulfate reduction to varying extents depending on community composition shaped by enrichment history. This was incubated in triplicate cultures of the two trained communities and the untrained community in diluted sewage sludge (COD ~1.5 g / L) with added gypsum. This design allowed us to assess the influences of enrichment history and microbial community on sulfate reduction and methane production in solutions with low organic loading.

[0074] Both trained communities reduced sulfate more rapidly than the untrained community (FIGs. 2A-2C), leading to increased alkalinity generation. Although sulfate reduction rates were comparable between Trained Community #1 and Trained Community #2, only Trained Community #2 did not produce methane. Dilution of the sewage sludge reduced methane levels across all communities compared to the concentrated sludge, but only Trained Community #2 exhibited both increased sulfide production and near-complete suppression of methane compared to Trained Community #1 (FIG. 3). These results suggest that enrichment history and community composition determine the partitioning of electrons in less concentrated sewage sludge.

[0075] Efficient removal of organic matter remains a central goal of wastewater treatment. In addition to measuring sulfide, methane and alkalinity concentrations, the degradation of organic matter was assessed by measuring the mass of volatile solids (VS) and quantified the concentration of calcium and alkalinity in the solution. VS removal is a key performance metric in wastewater treatment, as it reflects the extent of organic degradation and assesses the performance of the sludge treatment process53. The incubations were terminated once sulfide concentrations stopped increasing and became comparable across treatments, indicating the cessation of sulfate reduction. Both trained communities removed more VS than the untrained community (Table 2), consistent with enhanced microbial activity. These results suggest that communities enriched and trained to degrade sewage sludge (FIGs. 2A-2C) degraded organic matter more efficiently and released more alkalinity. Thus, the addition of inoculum can increase the rates and the efficiency of sludge treatment processes that release calcium into the solution.

[0076] Because sulfate reduction liberates calcium, it was assessed whether the alkalinity generated under these conditions was sufficient to drive carbonate precipitation during air drying of the entire culture solution (Table 2).

[0077] Table 2: Volatile solid (VS) removal under low organic loading.184229050. vl0050.2404001 (MIT-26176)

[0078] Microbes generated overall less alkalinity in the presence of low organic loading (FIG. 2C) than in medium with high organic loading (FIG. 8A) but released more calcium into the solution (FIG. 4B). Because calcium is released only when sulfate is reduced, carbonate formation depends primarily on sulfate reduction.

[0079] In the attempt to relate the differences in sulfate reduction, methane production, and the removal of volatile solids to the structure of microbial communities, the 16S rRNA gene of all enrichment cultures and the untrained community were sequenced (FIG. 5 and FIGs. 6A and 6B). This allowed us to compare microbial composition across treatments, with a focus on groups involved in fermentation, sulfate reduction, and methanogenesis. The communities sampled on day 17 were compared because sulfate reduction had ceased by that time in the two trained communities, but methanogenesis proceeded in one of the trained communities (FIGs. 2A-2C). These data provide a basis for evaluating the effect of enrichment strategies on the structure and function of microbial communities in the presence of low organic loading.

[0080] Both trained communities converged toward a similar taxonomic composition at the order level (FIG. 5), despite different enrichment histories. Community profiles were consistent across biological triplicates, indicating that selective pressures drove reproducible community structures even in the complex and variable background of sewage sludge. Thus, distinct enrichment processes repeated over time resulted in streamlined communities.

[0081] The abundances of different families changed as a function of enrichment procedures relative to the native sewage community. Chitinophagales was present at similar levels across all conditions, but the two trained communities contained more Desulfobacterales than the untrained community. Several known fermentative orders were abundant across all treatments, including Bacteroidales, Christensenellales and Peptostreptococcales- Tissierellales. Bacteroidales was the most abundant fermenter in both trained communities. Izemoplasmatales was also elevated in both trained communities relative to the untrained community. In contrast, Syntrophobacterales, Syntrophibac ter ales, Eubacterales, Flavobacterales, Fibrobacterales, and Pseudomonadales were reduced in194229050. vl0050.2404001 (MIT-26176)Trained Community #1 compared to the untrained community. Trained Community #2 contained lower abundances of Fibrobacterales, midas o 4, and Pseudomonadales relative to the untrained community. Pseudomonadales was highly abundant in the untrained community but nearly absent in both trained communities, Synergistales and Sphingomonadales were also more abundant in the untrained community. These results suggest that the enrichment procedure changed the fermentative community and the abundances of sulfate reducing bacteria while preserving a core community structure dominated by Bacteroidales.

[0082] To further relate these taxonomic shifts to the observed rates of sulfate reduction and methanogenesis, the relative abundances of sulfate reducing and methanogenic families were analyzed (FIGs. 6A and 6B). Trained Community #1 contained the 16S rRNA sequences of sulfate reducers and methanogens, with the latter sequences being about three times more abundant than in Trained Community #2 or the untrained community. Trained Community #2, by contrast, was enriched only in sulfate reducers compared to the untrained community. Desulfomicrobiaceae were present in both trained communities, but more abundant in Trained Community #2. Trained Community #1 was also enriched in Desulfosarcinaceae and contained fewer sequences of Desulfitobacteriaceae , Desulfobulbaceae. Desulfatiferula and Desulforegulaceae . Syntrophobacteraceae was nearly absent in Trained Community #1 but present in both Trained Community #2 and the untrained community. Trained Community #2 was further enriched in Desulfobacteraceae .Several other sulfate reducing families, including Desulfurisporaeceae. were detected only in trace amounts across all conditions. Methanogens exhibited low abundance across all treatments, but their community compositions changed with the treatment. The relative abundance of methanogens was the highest in Trained Community #1 (-0.6%) and dominated by Methanosarcinaceae and Methanomassiliicoccaceae . Trained Community #2 and the untrained community instead contained primarily Methanobacteriaceae (-0.2%). Methanosarcinaceae and Methanomassiliicoccaceae were enriched in Trained Community #1, whereas Trained Community #2 and the untrained community contained mostly Methanobacteriaceae . Trained Community #2 showed only a slight increase in Methanobacteriaceae and a decrease in Methanosarcinaceae relative to the untrained community. These results suggest that the electrons in Trained Community #2 reduced sulfate without being supplied to any abundant or metabolically versatile methanogens. These204229050. vl0050.2404001 (MIT-26176) findings show that enrichment strategies can reproducibly shift microbial communities in sewage sludge, shaping their metabolic fluxes under different loading conditions.DiscussionEnrichments and decreased organic loading improve carbon retention

[0083] The experiments of the present disclosure show that sulfate reducing microbial communities can effectively convert gypsum to carbonates and sulfide using municipal sewage sludge as electron donors. With appropriate enrichment cultures and environmental controls, microbial reduction of sulfate from gypsum can generate alkalinity with negligible methane emissions. Here it was shown that achieving effective sulfate reduction requires balancing microbial competition at different organic loading conditions. Although higher organic loadings allow gypsum bioconversion, they also divert electron donors toward methanogens, resulting in comparable rates of methane generation and sulfate reduction. Lower organic loading mitigates this competition and reduces methane emissions, but optimal enrichment is essential to achieve consistent methane suppression. The methane suppression observed here is consistent with other studies52 56 59but had not been quantified previously in gypsum-converting wastewater systems. The ability to control the partitioning of electrons toward methanogenesis or sulfate reduction and the generation of alkalinity requires a detailed understanding of microbial interactions that govern the competition for substrates and syntrophy.Syntrophy and competition for substrates

[0084] The composition and function of microbial communities enriched in the presence of sewage sludge and sulfate reflect selective pressures due to the availability of organic substrates and syntrophic interactions between fermenters and terminal reducers (methanogens or sulfate-reducing bacteria). As the most abundant organisms in the anaerobic communities, fermenters form the metabolic foundation that supports terminal metabolisms through the production of short-chain fatty acids (VFAs), hydrogen and alcohols60. Dominant fermentative orders observed here, Bacteroidales, Christensene Hales, and Peptostreptococcales- Tissierellales, are common in anaerobic digesters61and likely mediate the primary degradation of proteins and carbohydrates into acetate, propionate, butyrate, and lactate62 65. These VFAs are taken up by SRB and methanogens, and their availability directly influences competitive interactions and downstream community structure. Increased abundances of Bacteroidales and Izemoplasmatales in both trained communities indicate214229050. vl0050.2404001 (MIT-26176) enhanced hydrolysis and acidogenesis, selecting for efficient fermenters and suggesting that the structure of the fermentative community structure fundamentally constrains terminal electron accepting pathways. Typically, VFAs do not accumulate significantly in stable anaerobic digesters66, suggesting the close coupling of fermentation kinetics and subsequent terminal metabolisms.

[0085] Distinct taxonomic profiles of sulfate reducers and methanogens across communities reflect differences in substrate availability and metabolic preferences. In Trained Community #1, high methane production coincides with increased abundance of metabolically versatile methanogens, particularly Methanosarcinaceae and Methanomassiliicoccaceae . Methanosarcinaceae can utilize acetate, hydrogen, and methanol67, while Methanomassiliicoccaceae are obligate methylotrophic methanogens reliant on hydrogen-dependent reduction of methanol68. The increased abundances of these taxa in Trained Community #1 suggest that the availability of diverse fermentation byproducts favors methanogens capable of exploiting multiple substrates. In contrast, the most abundant methanogens in Trained Community #2 and the untrained community belong to the hydrogenotrophic family Methanobacteriaceae, which generates methane by oxidizing H269’70. The negligible methane production in these communities likely reflect limitation by H2 and competitive exclusion by sulfate reducers.

[0086] The decline in secondary fermenters — e.g., acetogens which convert the less competitive non-acetate VFAs to acetate and H2, such as Syntrophobacterales — in Trained Community #1 suggests rapid consumption of VFAs by terminal reducers, reducing the necessity for syntrophic oxidation of intermediates like propionate to acetate. The dominance of the sulfate reducing family Desulfosarcinaceae in Trained Community #1 provides a possible mechanistic explanation: these sulfate reducers preferentially oxidize substrates that are largely non-competitive with methanogens, such as propionate and certain aromatic compounds. This substrate partitioning may allow for the observed coexistence with acetoclastic methanogens such as Methanosarcinaceae that can metabolize acetate that SRB do not consume. Additionally, Methanomassiliicoccaceae, an obligate hydrogen-dependent methylotrophic methanogen68, are more abundant in Trained Community #1. This trend likely reflects the availability of hydrogen and methanol provided by fermenters and minimal competition of Methanomassiliicoccaceae and Desulfosarcinaceae for hydrogen. Although Desulfosarcinaceae can engage in syntrophy with anaerobic methane-oxidizing archaea (ANME)71, their co-occurrence here with abundant methanogens is likely due to224229050. vl0050.2404001 (MIT-26176) complementary, non-competitive substrate use. In contrast, Trained Community #2 contains primarily the acetate-oxidizing Desulfobacteraceae that directly compete with methanogens for this substrate. Early establishment of substrate-competitive SRB in Trained Community #2 thus may suppress methanogenic activity, shifting the microbial community toward sulfate reduction. This competition has increased importance due to the lower organic loading and thus increased organic substrate scarcity. These sulfate reducers are prevalent in the initial inoculum. These results highlight the importance of substrate competition among fermenters, methanogens, and sulfate reducers in the shaping of communities that mediate gypsum conversion using electron donors from sewage (FIG. 7).

[0087] FIG. 7 is a schematic showing the differences between Trained Community #1, Trained Community #2, and an Untrained Community (Baseline) of low organic loading 770a, 770b, and 770c (respectively) and high organic loading 771a, 771b, and 771c (respectively). Trained Community #1 is in a fermenter in both low and high organic loading 770a and 771a, that results in the formation of both sulfate reducing bacteria (SRB) and methane producing archaea (MPA), which produces hydrogen sulfide and methane respectively. Similarly, the Untrained Community also in a fermenter in both low and high organic loading 770c and 771a, that also forms SRB and MPA that produces hydrogen sulfide and methane, respectively. The difference between Trained Community #1 and the Untrained Community is that Trained Community #1 produces more or equivalent methane and more hydrogen sulfide. Trained Community #2 at high organic loading 771b behaves similarly to Trained Community #1 at high organic loading 771a. Trained Community #2 id different at low organic loading as it does not produce MPA, therefore it does not produce methane. In doing so, it selectively produces hydrogen sulfide through SRB.

[0088] Together, these experiments and observations support a model in which the balance between sulfate reduction and methanogenesis is driven not only by thermodynamic considerations, but also by competitive interactions among terminal reducers for the substrates produced by anaerobic fermenting microbes. The competitive interactions are increasingly more important with lower organic loading due to increased organic substrate scarcity, thus adding both an additional layer of complexity and opportunity for methane suppression in low organic anaerobic digesters. Enrichment protocols that select for the desired functions of microbial communities and the inoculation of enriched communities into sewage can ensure robust sulfate reduction rates and reduce the production of the potent234229050. vl0050.2404001 (MIT-26176) greenhouse gas methane, offering practical implications for controlling methane emissions during gypsum bioconversion and related anaerobic treatment processes.Scalability and Community Transferability

[0089] Nearly all dominant taxa in the trained communities were also detected in the untrained community, attributing the observed shifts to the enrichment of microbes that had been present in sewage rather than the recruitment from any external inoculum. Thus, the performance of enrichments likely reflects community changes shaped by environmental and historical conditions. Because the necessary organisms are already present in sewage sludge, in situ enrichments that favor sulfate reduction and minimize methanogenesis may be feasible in full-scale systems by applying selective pressures such as transient sulfide accumulation (sulfide shock), pre-starvation, or transient pH spikes to shift communities toward sulfate reduction.

[0090] Directing electron donors toward sulfate reduction without significant methane production could leverage existing wastewater treatment infrastructure. Volatile solid (VS) removal was higher for trained communities, but it remains unclear if VS removal will be sufficient in large anaerobic digesters with semicontinuous operation. As the community which produced methane did remove more VS in the same time frame (Table 2), a balance between partial methanogenesis and sulfate reduction may need implemented if future VS removal is insufficient. However, optimized flow-through systems may enhance VS removal and should be the focus of future work. While large dilutions of livestock manure are economically unfavorable for gypsum conversion30, municipal sewage may be a better option because it is often thickened 25-50 times before digestion. Avoiding this step, and instead adding gypsum earlier in the treatment process, could achieve the ~60x dilution that was explored by the current study. Alternatively, seawater presents a viable post-thickening diluent, providing additional sulfate and cations without significant disruption to existing wastewater infrastructure. A similar approach has been implemented in Hong Kong, where the SANI (Sulfate reduction, Autotrophic denitrification and Nitrification Integrated) process effectively treats sulfate-rich wastewater13 18. Similar approaches can be adopted for gypsum conversion and alkalinity generation.Conclusions and Outlook

[0091] The present disclosure demonstrated that an enriched microbial community (Trained Community #2) reduced sulfate from gypsum in batch cultures with minimal methane production in the presence of sewage sludge as the electron donor. Conditions244229050. vl0050.2404001 (MIT-26176) conducive to sulfate reduction included low organic loading and did not require the addition of external carbon sources. Compared to the untrained community, the enriched culture reduced sulfate faster, equivalent to 250 mg gypsum / L / day. Methane yields were similarly low in Trained Community #2 and the untrained community that was present in sewage. These results highlight the importance of enrichment history and selective pressure in controlling microbial competition: only enriched communities achieved high sulfate reduction without high rates of methanogenesis. Conversely, a different enriched community (Trained Community #1) produced much more methane than either Trained Community #2 or the untrained community. To achieve the low rates of methanogenesis, organic loading needed decreases to allow carbon substrate scarcity to drive syntrophy and competition. Future work should evaluate whether similar outcomes can be achieved through in-situ enrichment in continuous reactor systems, where selective pressures may be applied without the need for external inoculation, and if the extent of VS removal is sufficient in these systems.References

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[0163] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0164] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.314229050. vl

Claims

0050.2404001 (MIT-26176)CLAIMSWhat is claimed is:

1. A method for sequestering carbon dioxide, the method comprising: a) forming a mixture by combining a sulfate salt source with an organic matter source under anaerobic conditions; b) reacting at least one trained microbial community with the mixture in a manner that causes the sulfate salt source and organic matter source to be converted into hydrogen sulfide (H2S), sulfate salt cations, and bicarbonate ions (HCO3 ); and c) sequestering carbon dioxide as an insoluble carbonate salt by reacting the sulfate salt cations with the bicarbonate ions that forms the insoluble carbonate salt, carbon dioxide (CO2), and water.

2. The method of claim 1, further comprising separating the carbonate salt and hydrogen sulfide.

3. The method of claim 2, further comprising collecting the carbonate salt and repurposing same for industrial applications.

4. The method of claim 1, further comprising oxidizing the hydrogen sulfide in a manner that forms elemental sulfur (S°).

5. The method of claim 4, wherein the oxidizing is performed via precipitation as metal sulfides.

6. The method of claim 4, wherein the oxidizing is performed via microbiological metabolism.

7. The method of claim 4, wherein the oxidizing is performed via a biological or abiotic system.

8. The method of claim 1, wherein the at least one trained microbial community reduces sulfate ions without generating methane.324229050. vl0050.2404001 (MIT-26176)9. The method of claim 1, wherein the at least one trained microbial community does not produce methane when organic matter has a concentration ratio of organic matter source to sulfate salt source is about 0.9 to about 3.7.

10. The method of claim 1, wherein the organic matter source comprises at least one of organic waste, sewage, animal manure, compost waste, agricultural waste, food processing residues, papermill sludge, brewery waste, meat processing waste, wastewater, natural organic material, leaf litter, wood, com stalks, or any combination thereof.

11. The method of claim 1, wherein the sulfate salt source comprises industrial waste gypsum, phosphogypsum, flue gas desulfurization (FGD) gypsum, natural gypsum, titanium gypsum, anhydrite, barite, epsomite, celestinejarosite, calcium sulfate sources or anhydrides thereof, barium sulfate sources or anhydrides thereof, magnesium sulfate sources or anhydrides thereof, copper (II) sulfate sources or anhydrides thereof, lead (II) sulfate sources or anhydrides thereof, strontium sulfate sources or anhydrides thereof, or any combination thereof.

12. The method of claim 1, wherein the method is performed in a bioreactor.

13. The method of claim 12, wherein the bioreactor is an upflow anaerobic sludge blanket reactor, fluidized bed reactor, packed bed reactor, membrane bioreactor, sequencing batch reactor, or anaerobic digester.

14. The method of claim 1, wherein the method is performed underground.

15. The method of claim 1, further comprising concentrating the mixture and repeating (b) and (c).

16. A bioreactor compri sing : a) a multi-phase inlet to the bioreactor, the multi-phase inlet comprising a sulfate salt source and an organic matter source; b) at least one trained microbial community; and c) an anaerobic chamber within the bioreactor.

17. The bioreactor of claim 16, further comprising at least one container separating a carbonate salt, hydrogen sulfide (H2S), or a carbonate salt and hydrogen sulfide.334229050. vl0050.2404001 (MIT-26176)18. The bioreactor of claim 17, wherein the hydrogen sulfide is further oxidized to elemental sulfur within the at least one container separating carbonate salt, hydrogen sulfide, or carbonate salt and hydrogen sulfide.

19. The bioreactor of claim 16, wherein the at least one trained microbial community converts the sulfate salt source and the organic matter source into hydrogen sulfide (H2S), sulfate salt cations, and bicarbonate ions (HCO3 ), and the sulfate salt cations react with the bicarbonate ions forming an insoluble carbonate salt, carbon dioxide, and water.

20. The bioreactor of claim 16, wherein the organic matter source comprises at least one of organic waste, sewage, animal manure, compost waste, agricultural waste, food processing residues, papermill sludge, brewery waste, meat processing waste, wastewater, natural organic material, leaf litter, wood, com stalks, or any combination thereof.

21. The bioreactor of claim 16, wherein the sulfate salt source comprises industrial waste gypsum, phosphogypsum, flue gas desulfurization (FGD) gypsum, natural gypsum, titanium gypsum, anhydrite, barite, epsomite, celestinejarosite, calcium sulfate sources or anhydrides thereof, barium sulfate sources or anhydrides thereof, magnesium sulfate sources or anhydrides thereof, copper (II) sulfate sources or anhydrides thereof, lead (II) sulfate sources or anhydrides thereof, strontium sulfate sources or anhydrides thereof, or any combination thereof.

22. The bioreactor of claim 16, wherein the bioreactor is an upflow anaerobic sludge blanket reactor, fluidized bed reactor, packed bed reactor, membrane bioreactor, sequencing batch reactor, or anaerobic digester.

23. A method for producing trained microbial communities, the method comprising: a) creating a seed by collecting at least one microbial community; b) culturing the at least one microbial community collected to produce a seed community; c) training the seed community for converting a sulfate salt source and organic matter to alkaline solutions and sulfidic solutions to produce a trained seed community; and344229050. vl0050.2404001 (MIT-26176) d) selecting a trained microbial community from the trained seed community.

24. The method of claim 23, wherein the trained microbial community reduces sulfate ions without generating methane.

25. The method of claim 23, wherein the trained microbial community does not produce methane when the concentration ratio of organic matter source to sulfate salt source is about 0.9 to about 3.7.

26. The method of claim 23, wherein the trained microbial community is enriched by applying a selective pressure.

27. The method of claim 23, wherein the trained microbial community comprises sulfate reducing bacteria, fermenting bacteria that produce substrates for sulfate reducing bacteria and a reduced contribution of hydrogenotrophic methanogenic archaea relative to the untrained communities.

28. The method of claim 27, wherein the sulfate reducing bacteria comprise more Desulfomicrobiaceae, Desulfurisporaeceae, Desulfobacterales, or a combination thereof, and fewer Me thanobacteriaceae and Methanomassiliaceae than untrained microbial communities.

29. The method of claim 23, wherein collecting at least one microbial community is from sediments.354229050. vl

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