Methods and systems for treating fluid with vacuum-assisted anaerobic digestion

Abstract

Methods and systems are described for controlling a vacuum-assisted anaerobic digester to optimize microbe populations and to recover ammonia. The organic loading rate of the digester (OLR) can be controlled over a sufficient duration so that the population of methanogens transition so that they become predominantly Methanosarcinaceae, which are faster growing methanogens. Ammonia recovery can be accomplished by stripping ammonia and carbon dioxide out of the fluid in the digester and condensing the evaporate in the presence of water to form ammonia carbonates.

Description

METHODS AND SYSTEMS FOR TREATING FLUID WITH VACUUM-ASSISTEDANAEROBIC DIGESTION TECHNICAL FIELD

[0001] This disclosure relates generally to treating wastewater fluids in a vacuum-assisted anaerobic digester to transform the methanogen microbe population in the fluid, andto recover ammonia from the digestate.BACKGROUND

[0002] One of the leading sludge management technologies is anaerobic digestion.Anaerobic digestion (AD) is a promising technology that can convert the organic wastes to either volatile fatty acids or methane in a multi-step biochemical process in which the wastes are broken down by anaerobic microorganisms in an oxygen-free environment. The basic steps of anaerobic digestion are hydrolysis, acidogenesis, acetogenesis, and methanogenesis.

[0003] In the first step, hydrolysis, hydrolytic bacteria degrade the complex organicpolymers such as proteins, carbohydrates, and lipids into soluble monomers. In waste- activated sludge (WAS), a major part of the organic compounds is bordered in a polymeric network formed by extracellular polymeric substances (EPSs). EPSs are highly hydrated structures with importance in bio flocculation, settling, and dewatering the sludge. EPS in WAS is mainly attributed to the proteins and carbohydrates which need to be disintegrated to make the intracellular content available to the microorganisms.

[0004] The second step is acidogenesis, for example fermentation, where theproducts of hydrolysis further degrade to form volatile fatty acids (VFA) such as acetic acid, propionic acid, butyric acid, iso-butyric acid, valeric acids, and the like, ammonia, hydrogen sulfide, carbon dioxide, and other by-products.

[0005] The next step, acetogenesis, involves acetogenic bacteria which convertorganic acids into acetic acid, hydrogen, and carbon dioxide.

[0006] The final stage of anaerobic digestion is methanogenesis, whereinbiomethane is produced by two groups of methanogenic organisms: acetoclastic methanogens, which degrade acetate into methane and carbon dioxide, and hydrogenophilic methanogens, which use hydrogen as an electron donor and carbon dioxide as an acceptor to produce methane.Confidential - Company Proprietary

[0007] AD thus treats and stabilizes the sludge and recovers value-added productsin the form of methane or hydrogen and volatile fatty acids (VFA) through fermentation. The VFA recovered from the fermented sludge can be used for several applications such as a carbon source for biological nutrient removal on-site, biodegradable plastics production, and hydrogen production. AD has many other benefits, such as solid reduction, decreasing greenhouse gas emissions, odor reduction, and increasing non-market benefits compared to the other waste treatment technologies.

[0008] Some of the challenges associated with AD include long hydraulic retentiontimes of the soluble fraction of a fluid (HRT) and low methane and VFA yields. Hydraulic retention time and loading rate of the AD dictate the volume of the digester that need to be designed and built. Vacuum-assisted anaerobic digestion processes have been proposed todecouple the hydraulic retention time of the soluble fraction from the residence time of thesolids fraction of the fluid being treated. This technology can also enhance methane and / orhydrogen production. Suitable vacuum-assisted processes and systems are described in U.S.Patent Application No. 17 / 742,905 and U.S. Application No. 18 / 199,548, the entiredisclosures of which are incorporated by reference herein. Additional improvements in thevacuum-assisted anaerobic digestion are desired to optimize the operation of the AD andmaximize recovery of resources. SUMMARY

[0009] Aspects of this disclosure provide improved vacuum-assisted anaerobicdigestion that can optimize the methanogenic microbe population to promote methanogenesis,and concurrently recover methane and ammonia from wastewater fluids.

[0010] In one aspect, the invention includes a method for treating a fluid thatincludes a particulate fraction and a soluble fraction. The method includes (i) feeding thefluid to an anaerobic digester; (ii) physically and biochemically transforming solids in theparticulate fraction of the fluid with microbes in the anaerobic digester, including apopulation of methanogens; (iii) subjecting at least a portion of the fluid that has beenbiochemically treated in the anaerobic digester to a vacuum pressure to evaporate off at leasta portion of the soluble fraction of the fluid as evaporate and to provide a thickened fluid; (iv)further treating the thickened fluid in the anaerobic digester so that it is further physically andbiochemically transformed; and (v) controlling at least one parameter, including an organicloading rate (OLR) in the anaerobic digester over a sufficient duration, so that the populationof methanogens transition to predominantly Methanosarcinaceae.Confidential - Company Proprietary

[0011] In another aspect, the invention includes a method for treating a fluid thatincludes a particulate fraction and a soluble fraction. The method includes (i) feeding the fluid to an anaerobic digester; (ii) physically and biochemically transforming solids in the particulate fraction of the fluid with microbes in the anaerobic digester; (iii) subjecting at least a portion of the fluid that has been biochemically treated in the anaerobic digester to a vacuum pressure to evaporate off at least a portion of the soluble fraction of the fluid as evaporate and to provide a thickened fluid, and where the evaporate includes carbon dioxide and ammonia; (iv) further treating the thickened fluid in the anaerobic digester so that it is further physically and biochemically transformed; and (v) condensing at least a portion of the evaporate in the presence of a sufficient amount of water to provide a condensate in which at least some of the ammonia forms ammonium and at least some of the carbon dioxide formscarbonic acid to cause at least one of ammonium carbonate and ammonium bicarbonate to beformed in the condensate. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Fig. 1 is a schematic diagram of a vacuum-assisted anaerobic digestionsystem;

[0013] Fig. 2 is a graph showing the production of methane, VFA, and COD in avacuum-assisted anaerobic digestion process, as well as a change in methanogen population; and

[0014] Fig. 3 is a graph showing the results of an experiment to quantify therecovery of ammonia in the condensate of a vacuum-assisted anaerobic digester. DETAILED DESCRIPTION OF EMBODIMENTS

[0015] Aspects of the methods and systems described herein include controlling theorganic loading of a vacuum-assisted anaerobic digester to optimize the biochemical process,and to improve the recovery and use of ammonia from the treated fluid. The feed to theanaerobic digester can include any type of biosolids, e.g., wastewater treatment biosolids(primary and biological treatment biosolids), organic fraction of municipal solid wastes, food wastes, organic industrial wastes, and agricultural wastes e.g. animal manure, bioethanol industry wastes, etc., and includes soluble and particulate organics. The feed may also include ionic fluids that have been used to extract contaminants or valuable components.

[0016] The fluid that is treated is a contaminated mixture, which includes a solublefraction and a particulate fraction. The contaminated mixture is subjected to anaerobicConfidential - Company Proprietarydigestion and subjected to vacuum pressure to produce an evaporated portion and a thickenedfluid. In one aspect, the contaminated mixture can be fed to a vacuum-integrated reactor, e.g., a digester with a vacuum pump associated therewith for applying a vacuum to the vacuum-integrated reactor, in an in-situ method. In another aspect, the contaminated mixture can befed to an anaerobic digester, and a portion of the digestate can be separately fed to a vacuum-integrated side stream treatment unit in an ex-situ method. As described in the abovereferenced applications, by using a vacuum pressure with a digester, the retention time of the non-volatile particulate fraction of the fluid (solids retention time or "SRT") can bydecoupled from the retention time of the soluble fraction of the fluid ("HRT") in useful ways.

[0017] Controlling Microbial Population

[0018] In connection with this invention, it was discovered that, in vacuum-assistedanaerobic digestion, the methanogen population can be advantageously manipulated by controlling at least one parameter, which includes the organic loading rate (OLR) to the digester for a sufficient duration. For example, Methanosaeta is generally the predominantmethanogen in an anaerobic digester. But, by controlling the OLR and optionally otherparameters, the methanogen population in the digester can be induced to change from apredominantly Methanosaeta population to a predominantly Methanosarcinaceae population.For example, by sustaining OLRs in a range of greater than 2.5 kg / VS / m3 / d, or from 3 to 30 kg / VS / m3 / d, or from 3.5 to 7.5 kg / VS / m3 / d, where "VS" is volatile solids, theMethanosarcinaceae population in the digester can become the dominant methanogen (themost populous methanogen). In some aspects, after the change in microbe population is induced, the Methanosarcinaceae population can be more than 50% of methanogens in the digester or more than 60%, for example, and the Methanosaeta population can be less than10% of the methanogens in the digester or less than 5%, for example. The OLR can also beexpressed based on chemical oxygen demand (COD), and by sustaining OLRs in a range ofgreater than 3 kg / COD / m3 / d, such as from 5 to 50 kg / COD / m3 / d, or from 5.5 to 25 kg / COD / m3 / d, the Methanosarcinaceae population in the digester can become the dominant methanogen. The duration of the sustained high OLRs required before the methanogen population changes in this manner can be greater than 5 days, such as greater than 10 days, greater than 20 days, or greater than 30 days, for example. Alternatively, the duration of the sustained high OLR can be greater than 1 turnover of the mean SRT, or greater than 2 turnovers of the SRT, or 3 turnovers of the SRT, for example.

[0019] It is believed that high OLRs contribute to shifting the methanogen microbepopulation to Methanosarcinaceae population since the organic food source changes, andConfidential - Company Proprietarymay contribute to relatively higher amounts of organic acids, such as propionic acid and butyric acid, that are produced through hydrolysis.

[0020] Other variables may also need to be concurrently controlled to facilitate theshift of this methanogen microbe population such as the ratio of the solids retention time(SRT) to the hydraulic retention time (HRT), and the stripping rate of ammonia, for example.The vacuum-assisted digester can be controlled so that a ratio of the SRT to HRT is greater than 2, such as from 3 to 15 or from 4 to 8.

[0021] The concurrent stripping of ammonia from the digester via the vacuumprocess may facilitate change of the methanogen population since ammonia is a toxin in thedigester that inhibits the growth of most microbes. Thus, the amount of ammonia that isstripped from the digester as evaporate can be controlled, e.g., by controlling vacuumpressure and temperature, to further induce a shift of the methanogen microbe population toMethanosarcinaceae population. The impact of the vacuum stress itself, and the vacuum pressure and the duration of vacuum pressure that is selected, may also affect the microbialculture, in particular methanogens (acetoclastic and hydrogenotrophic) which are consideredamong the most vulnerable microbial communities in the anaerobic process.

[0022] In some embodiments, the digester can be inoculated with a dose ofMethanosarcinaceae (e.g., a digestate or fluid in which Methanosarcinaceae represents at least 20% or at least 50% of the methanogen population) to promote dominance of that strain in the digester.

[0023] Additionally, in some embodiments, the digester may be inoculated withhydrolytic bacteria capable of efficient lignocellulose degradation, including extremophiles such as Caldicellulosiruptor bescii. This organism is a thermophilic, cellulolytic bacterium known for its robust enzymatic arsenal, including modular cellulases such as CelA, which can cleave both crystalline and amorphous cellulose, as well as hemicellulose, without theneed for exogenous enzymatic supplementation. C. bescii also tolerates elevated temperatures(up to 80°C) and performs optimally under anaerobic and mildly acidic to neutral conditions, making it particularly well-suited for use in high-solids, thermophilic, or electrochemically enhanced digesters.

[0024] Inoculating with C. bescii, either in pure culture or as part of a thermophilichydrolysate-producing consortium, can accelerate the rate-limiting hydrolysis step by initiating rapid depolymerization of cellulose, xylan, and other plant-derived polysaccharides. This results in enhanced solubilization of organic matter and increased availability offermentable intermediates (e.g., sugars, lactate, acetate, and hydrogen) that feed downstreamConfidential - Company Proprietarysyntrophic and methanogenic pathways. Furthermore, the simultaneous enrichment ofMethanosarcinaceae supports rapid conversion of those intermediates to methane,particularly under dynamic or intensified loading regimes.

[0025] Controlling the digester to promote Methanosarcinaceae dominance can beadvantageous because Methanosarcinaceae has high growth rates, is more robust toward pH changes, is more tolerant to ammonia, and is more resistant to organic shock loading and high VFA concentrations as compared to Methanosaeta. This allows the vacuum-assisteddigestion process to be run under more optimal conditions for methanogenesis with shorterretention times.

[0026] The co-occurrence of syntrophic microbes and methanogens also facilitatesperformance of an anaerobic digester as their interactions can advance thermodynamicallyunfavorable reactions, such as propionate and butyrate oxidation. The operation of vacuum- assisted digester to provide a predominant methanogen population of Methanosarcinaceaealso can cause or be associated with possibly advantageous increases in certain syntorphssuch as Clostridium_sensu_stricto_1, Pelotomaculum, Syntrophomonas, Mestoga, DMER64, Synergistaceae, Smithella, and Syntrophobacher.

[0027] Recovery

[0028] In one aspect, the inventors discovered that ammonia can be stripped fromthe treated fluid and recovered in condensate of the evaporated fraction as ammoniacarbonate and / or ammonia bicarbonate.

[0029] During the vacuum-assisted digestion process, carbon dioxide in the digestercan be first stripped and removed in the evaporate, which will cause the pH of the digestate toincrease since carbon dioxide is acidic. As the pH rises, the concentration of free ammonia in the digestate will increase, and ammonia in the digestate will be stripped and removed in the evaporate, which will occur concurrently with the stripping of carbon dioxide for at least some period. The vacuum-assisted digester can be controlled so that the pH of the digestatetreated by the vacuum evaporation unit increases to at least 7.25, such as from 7.5 to 8.5, orfrom 8.5 to 9.5, to allow a sufficient amount of ammonium to shift from ionized to un-ionizedform, hence from ammonium ion towards ammonia gas, hence to a form which could bestripped from the digester. For example, at a pH of 9.25 about 50% of the ammonia exists asammonia gas in the digester. Ammonia can be stripped from the digester in this way without adding pH adjusting agents (e.g., a base) to the digester.

[0030] When the evaporate is cooled in the presence of a sufficient amount of water,the carbon dioxide will form carbonic acid according to the following equation:Confidential - Company Proprietary

[0031] CO₂(g) + H₂O ⇌ H₂CO₃ ⇌ HCO₃⁻ + H⁺ ⇌ CO₃²⁻ + 2H⁺

[0032] And, in the presence of a sufficient amount of water, the ammonia will formammonium according to the following equation:

[0034] Depending on the concentrations of carbonic acid and ammonium in thecondensate, as well as other parameters as described below, the following reactions canoccur:

[0035] NH₃ + CO₂ + H₂O NH₄HCO₃ (ammonium bicarbonate); and

[0036] 2NH₃ + CO₂ + H₂O (NH₄)₂CO₃ (ammonium carbonate).

[0037] Concentrations of the ammonium and carbonic acid in the condensate can becontrolled based on the stripping process. Other parameters such as pH, temperature, and water availability can be controlled to promote the above reaction. For example, higher ammonia and carbon dioxide concentrations in the evaporate favor formation of ammoniacarbonate, lower temperatures favor condensation and solubility, a higher pH promotesammonia solubility, and the presence of carbon dioxide lowers the pH and promotesammonium formation. The presence of a water phase is also needed for dissolution and reaction.

[0038] In some embodiments, a separate carbon dioxide source can be supplied tothe condensate (e.g., by bubbling CO2 through the condensate), which will both add carbonicacid reactant to the condensate and lower the pH. Likewise, in some cases, a pH adjustingagent (e.g., acidic reagent) other than CO2 can be added to the condensate to the control thepH. When pH is adjusted in the condensate, the condensate can be acidified to a pH of lessthan 8 which facilitate the ammonia being trapped as ammonium salt. At those conditions, the strippable form of ammonia (i.e., ammonia gas) is minimal hence it can be captured well in the solution.

[0039] The ammonium salts are stable and allow the ammonia to be efficientlyrecovered in the condensate, thereby reducing the need to capture or otherwise manage theammonia in the gas phase. In this regard, in the absence of carbon dioxide, ammonia will tend to remain in the gas phase and is more difficult to capture in the condenser, particularly if the pH is high and no acid (including carbon dioxide) is present.

[0040] In some embodiments, a chlorine source can be combined with theammonium-containing condensate to react with ammonium and form chloramine. The chlorine source can include chlorine gas, hypochlorite, or other suitable oxidants capable of generating monochloramine, dichloramine, or trichloramine depending on pH and reactionConfidential - Company Proprietaryconditions. The resulting chloramine not only serves to chemically bind and control ammonia emissions during stripping or condensation but may also be utilized beneficially downstream within the treatment process.

[0041] In certain embodiments, chloramine formed in situ or added externally canserve as a disinfection agent for either the treated wastewater or the residual sludge. When applied to the sludge phase, chloramine can contribute to pathogen reduction sufficient to meet Class A biosolids standards under U.S. EPA 40 CFR Part 503 or equivalent international criteria. The use of chloramine in this context offers operational advantages over free chlorine or ozone, including greater stability, longer-lasting disinfecting power, and reduced formation of regulated disinfection byproducts.

[0042] Moreover, in additional embodiments, the oxidative potential of chloraminecan be harnessed to partially oxidize extracellular polymeric substances (EPS) present in the digested sludge matrix. EPS—primarily composed of proteins, polysaccharides, and humic substances—are known to interfere with sludge dewatering by retaining bound water and increasing sludge viscosity. Controlled chloramination of digested sludge may disrupt the structural integrity of EPS, leading to the release of intracellular water and enhanced floc disintegration, thereby improving the mechanical dewaterability of the sludge in downstream operations such as centrifugation, belt pressing, or vacuum filtration.

[0043] Sludge dilution with chloramine-rich condensate as well as the partialbreakdown of EPS and associated colloidal structures through chloramine-mediated oxidation can also result in a measurable reduction in the apparent viscosity of the sludge. This viscosity reduction enhances fluidity and pumpability, particularly under thermophilic or high-solids conditions, thereby reducing energy requirements for mixing and transfer, and improving heat and mass transfer within the digester or post-digestion handling systems. Lower viscosity also improves process controllability and enables more consistent operation of thickening and dewatering equipment. The rheological improvement observed is particularly beneficial in systems operating under intensified modes, such as those employing thermal hydrolysis, electrochemical conditioning, or vacuum-enhanced digestion, where baseline viscosities may otherwise limit throughput and energy efficiency.

[0044] Accordingly, the integration of chloramine chemistry within the broaderammonia and biosolids management strategy provides a multifunctional benefit: (i) nitrogen stabilization and ammonia odor control, (ii) enhanced hygienization of biosolids, and (iii) improved sludge rheology and dewatering performance. These effects are particularlyConfidential - Company Proprietarysynergistic in systems that incorporate intensified digestion, thermal hydrolysis, or electrochemical preconditioning, as disclosed elsewhere in this invention.

[0045] Additionally, stripping and recovering ammonia in this manner will reducethe overall emission of NOx from the system, in particular nitrous oxide (N2O). In this regard, the thickened digestate from the digester that is sent to waste or further dewatered will have less ammonia, and thus any centrate water recovered from the dewatering processes and recycled to the mainstream wastewater treatment system will have about 50%-60% less ammonia that can form NOx gases.

[0046] Vacuum-assisted Anaerobic Digester System

[0047] Fig. 1 is a schematic diagram of a vacuum-assisted anaerobic digestionprocess in which a separate vacuum unit is used to apply vacuum pressure in the ex-situmethod described above. A fluid 110 to be treated is sent to anaerobic digester 120 in which anaerobic microbes transform the fluid into digestate 130.

[0048] A portion 140 of the digestate is sent to a vacuum unit 160 which appliesvacuum pressure to the digestate to produce evaporate 165 and a thickened fluid 170. Atleast a portion thickened fluid 170 is recycled to be further treated in the anaerobic digester120.

[0049] A portion 150 of the digestate can be sent to a dewatering unit 230 (e.g.,centrifuge to further dewater the digestate. The dewatered solids 235 can be sent to waste or other downstream processes. The separated water (centrate) 240 can be recycled to the vacuum unit 160 or can be sent to downstream processes.

[0050] The evaporate 165 can be sent to a condenser where the evaporate is cooledin the presence of water (additional water can be added if needed) to provide a condensate 190. As indicated above, optionally carbon dioxide or another acidic reagent 185 can beadded to the condensate in the condenser 180 to promote the formation of ammoniumcarbonates. The condensate 190 can also optionally be combined with a chlorine source 210in tank 200 to form chloramine in a reactant stream 220. The chloramine-containing reactant stream 220 can be used as a disinfectant.

[0051] Example 1

[0052] Three anaerobic digesters were operated at an SRT of 20 and 30 days, oneconventional AD was used the control at an OLR of 1.1 ± 0.1 kg VS / m3 / d and SRT of 20days, and two other digesters were operated with vacuum intensification at OLR of 4.5 ± 0.4and 3.8 ± 0.4 kg VS / m3 / d. The intensification factors (IF), defined as SRT / HRT for the twovacuum-assisted anaerobic digesters were 4 (SRT of 20 days) and 5 (SRT of 30 days),Confidential - Company Proprietarycorresponding to HRTs of 5 and 6 days, respectively. The two vacuum-assisted anaerobicdigesters were operated to maximize the OLR.

[0053] The digesters were laboratory-scale digesters each having a total volume of2 L (1.8 L 232 working volume + 0.2 L headspace). The digesters were operated atmesophilic conditions (36-38 ℃) by using a wool heating jacket. Vertical stirrers, with fourpaddles were deployed for mixing at 200 rpm so the digestate would be homogenous. Thevacuum unit used was a rotary vacuum evaporation unit consisting of a chiller, vacuum pump, water 2bath, 2 L evaporator flask, and condensation flask. The entire system was maintained at 58 mbar, so evaporation takes place at mesophilic temperature (boiling temperature of 35 ℃).

[0054] Primary and thickened waste activated sludge (1:1 v / v) was the feedstock forthe digesters. Sludge was collected from a local wastewater treatment plant. The feed was 3.1% TS with a nitrogen content of 6.8% of VSS.

[0055] The operating condition of the digesters is shown in the table below.Table 1

[0056] Methanogenic activity (SMA) tests were conducted to assess acetoclasticmethanogenic activity. The test was conducted using an Automatic Methane Potential TestSystem (AMPTS) utilizing 400 mL liquid volume digesters (AMPTS_II, Bioprocess Control, Lund, Sweden) maintained at pH 6.9–7.2 and 37–38℃. CO2 in the off-gas was trapped with a 3 M NaOH solution and subsequently, methane generation was measured using a wet-tip meter. Acetic acid was used as a readily biodegradable substrate at 2 g / L (~ 0.85gCOD) and 0.8 gVSS was employed as inoculum. The inoculum was sourced from the individual Confidential - Company Proprietarydigesters to allow for a comparison of the methanogenic activity of the biomass in the vacuum-assisted anaerobic digesters with that of a conventional digester. The maximum specific methane production rates (MSMPRs) were calculated by normalizing the maximum net methane production rate to the inoculum VSS.

[0057] Biomethane potential (BMP) tests were conducted to evaluate overallhydrolysis-methanogenesis kinetics. The BMP tests were designed at an ISR of 1 (inoculummass to substrate VS mass ratio), using the mixture of PS / TWAS 50 / 50 v / v as the substrate and IF1, IF4, and IF5 digestate as the inoculum. Tests were conducted in triplicate and blank tests containing only inoculum were run in parallel. Like SMA, AMPTS was used for BMP test to run the batch reactors. The net methane production was calculated by subtracting theblank methane production from that in test samples. MSMPRs were normalized to gVSSinoculum to compare the activity of the various digestates.

[0058] Variation in the microbial community was analyzed by taking samplesduring start up phase (0-28 d), declining performance phase (29- 40 d), recovery phase (41 -51 d), and steady state phase (52-132 d). Sample preparation and DNA extraction was conducted on a uniform volume of 0.5 mL from feedstock and 0.25 mL of IF4 and IF5 using the FastDNA Spin Kit for Soil Extraction Kit (MP Biomedicals, Solon, OH, USA). The spectrophotometric method was used to determine the quality and amount of extracted genomic DNA by measuring the absorbance at wavelengths of 260 and 280 nm and calculating the A260 / A280 ratio. Amplification of the V4 regions of archaea and bacteria 16SrRNA was performed using the primer sequences of the specific regions

[0039] . A two-stepdouble-index PCR (PCR) method specifically designed for the Illumina MiSeq sequencing platform was performed. The amplicon libraries were sequenced in paired-end format with a reading of 300 bases, 2 x 300 base pairs on each side of the DNA strand on Illumina MiSeq at the genomic analysis platform, IBIS of Université Laval (Quebec, Canada). qPCR was employed to detect total archaea / bacteria with primers eub338 / eub518

[0040] . OriginPro software was used to create a heatmap of microbial abundance.

[0059] Table 2 below shows the steady-state parameters of the conventionaldigester (IF1) and the vacuum-assisted digesters.Confidential - Company ProprietaryTable 2

[0060] Fig. 2 shows that after the second SRT turnover from start-up (about 40 d),the performance of the digesters deteriorated as demonstrated by the reduction in methaneproduction. As shown in Fig. 2, by manipulating the ORL and the ammonia stripping in thevacuum-assisted digesters, the methane production was recovered after this time, and therewas a shift in the digesters from acetoclastic Methanosaeta to acetoclastic / hydrogenotrophicMethanosarcinaceae. After the digesters reached steady state, the Methanosaeta populationwas approximately 1% of the total methanogens and the Methanosarcinaceae constituted63.3% of the total methanogens.

[0061] Figure 2 also shows the yields of VFA and SCOD of the anaerobic digesters.As can be seen, SCOD and VFAs increase significantly during the period in which methane yield decreased.

[0062] Example 2

[0063] In Example 2, the efficiency of ammonia and VFA stripping was evaluatedunder different total dissolved solids (TDS) concentrations in an 80 L evaporator.Ammonium chloride, sodium acetate and sodium chloride were used to represent ammonia, acetate and TDS, respectively. Sodium hydroxide was added to manipulate the initial pH ofthe liquid in the evaporator to around 8. The test was done on three different days, in whichevery day included multiple runs. The amounts of chemicals added each day are summarized in Table 3, below.Table 3 - Initial operating conditions

[0064] For each test, the evaporator was filled up with clean water, then chemicalswere added, and then the vacuum evaporation was performed for 30 min at 35 °C for each run. Around 8 L of water evaporated each run. Therefore, 8 L of clean water was added to fix the evaporator volume at 80 L as an initial volume for each run. Samples were taken from the Confidential - Company Proprietaryevaporator and condensate tank each run. Ammonia was measured in the condensate and thewater in the evaporator for each run to evaluate striping efficiency. The results are shown inFig. 3.

[0065] Fig. 3 shows the total ammonia stripped, ammonia collected in condensateand the ratio of the ammonia in condensate to ammonia stripped. The ammonia collected in the condensate was in the range of 46-65% of the total ammonia stripped from the evaporator. This insufficient adsorption of ammonia in condensate could be related to absence of CO2 in the water, in which there was no in-situ acid (like carbonic acid) to protonate NH3 into non- volatile NH4+, resulting in poor capture efficiency. In contrast, anaerobic sludge typically contains relatively high levels of dissolved CO2 (as bicarbonate and carbonate), which, whenstripped, not only elevates the pH of the sludge to enhance NH3 release but also providesCO2 availability for reabsorption and in-condensate acidification. This buffering can create the conditions needed for stable ammonia capture in the condensate, significantly improvingrecovery efficiency without external chemicals. As indicated above, a separate CO2 sourcecan also be added to the condensate to promote ammonia capture.

[0066] Conclusion

[0067] As described above, the vacuum-assisted anaerobic digestion process can becharacterized by the application of strategic process intensification principles. The vacuum-assisted digestion can maintain a low hydraulic retention time (HRT), typically between 4 to 6 days, in conjunction with a longer solids retention time (SRT) exceeding 20 days. This configuration enables high organic loading rates (OLR), promotes selective microbial enrichment, and increases system throughput without compromising stability. By decoupling SRT and HRT through the selective removal of the soluble fraction via vacuum evaporation, the vacuum-assisted anaerobic digestion can provide a highly resilient microbial ecosystem and sustained bioconversion efficiency.

[0068] The vacuum-assisted anaerobic digestion system can also leverage thesimultaneous removal of ammonia and carbon dioxide under vacuum conditions to promote a favorable pH shift within the digester. As carbon dioxide is stripped, the alkalinity of the digestate increases, driving the ammonium-ammonia equilibrium toward free ammonia, which can be efficiently captured without external pH adjustment in some embodiments. The evaporate, enriched in NH₃ and CO₂, is subsequently condensed to yield an ammonium-rich condensate. In the presence of water, this leads to the spontaneous formation of ammonium carbonate and / or ammonium bicarbonate. The resulting condensate can be valorized directly Confidential - Company Proprietaryas a liquid nitrogen fertilizer or reacted with a chlorine source to generate chloramine fordisinfection purposes, supporting circular economy principles.

[0069] Vacuum intensification also creates conditions that favor a microbialtransition from Methanosaeta to Methanosarcinaceae. This shift is sustained by operating thedigester at elevated OLRs (typically above 3.5 kg VS / m³ / day) for a duration sufficient toexceed two complete SRT turnovers. The Methanosarcinaceae genus offers operationaladvantages due to its higher growth rate, greater tolerance to pH and ammonia fluctuations,and ability to metabolize a broader range of substrates including acetate and hydrogen. As a result, the digester becomes more resilient to load shocks and exhibits enhanced methane production kinetics, with volumetric methane yields exceeding 1.1 m³ CH₄ / m³-digester / day under steady-state conditions.

[0070] In addition to methanogenic adaptation, the vacuum-assisted anaerobicdigestion system supports the proliferation of beneficial syntrophic communities such asClostridium_sensu_stricto_1, Syntrophomonas, Pelotomaculum, and Mesotoga. These syntrophs enable thermodynamically challenging reactions such as propionate and butyrate oxidation, which are otherwise rate-limiting in conventional anaerobic digestion. The co- occurrence and enrichment of these microbial partners under vacuum-assisted, high-load conditions accelerate the stabilization of volatile fatty acids (VFAs) and enhance overall system robustness. This synergistic microbial structuring is a significant advantage

[0071] The condensate produced in this process contains ammonium concentrationssuitable for use as a feedstock in chloramine production or as a pre-fertilizer solution. Depending on operating parameters such as stripping intensity, feed nitrogen content, and condensate volume, ammonium concentrations in the range of from 500 to 5000 mg N / L orfrom 1000 to 3000 mg N / L can be achieved. This high-value liquid stream can eliminate theneed for additional nitrogen inputs in many applications and significantly reduces the nitrogen return load to mainstream wastewater treatment, thereby mitigating downstream nitrous oxide emissions.

[0072] The vacuum-assisted anaerobic digestion process also enables flexibleintegration into existing wastewater treatment infrastructure. By retrofitting conventional digesters with vacuum modules and modifying the operational control strategy, facilities can transition to an intensified regime without full infrastructure replacement. The technology provides a scalable pathway for utilities to enhance biogas recovery, reduce nutrient discharges, and move toward net-zero emissions targets. Confidential - Company Proprietary

[0073] Although specific embodiments were described herein, the scope of theinvention is not limited to those specific embodiments. The scope of the invention is defined by the following claims and any equivalents therein. As will be appreciated by one skilled in the art, aspects of the present disclosure may be embodied as an apparatus, system or method. Confidential - Company Proprietary

Claims

WHAT IS CLAIMED IS 1. A method for treating a fluid that includes a particulate fraction and asoluble fraction, the method comprising: feeding the fluid to an anaerobic digester; physically and biochemically transforming solids in the particulate fraction of thefluid with microbes in the anaerobic digester, including a population of methanogens;subjecting at least a portion of the fluid that has been biochemically treated in theanaerobic digester to a vacuum pressure to evaporate off at least a portion of the solublefraction of the fluid as evaporate and to provide a thickened fluid; further treating the thickened fluid in the anaerobic digester so that it is furtherphysically and biochemically transformed; and controlling at least one parameter, including an organic loading rate (OLR) in theanaerobic digester over a sufficient duration, so that the population of methanogens transitionto predominantly Methanosarcinaceae.

2. The method of claim 1, wherein the population of methanogens transitionfrom being predominantly Methanosaeta to being predominantly Methanosarcinaceae.

3. The method of claim 1, wherein controlling the at least one parameterfurther comprises controlling an amount of ammonia that is evaporated from the fluid as partof the soluble fraction of the fluid so that the population of methanogens transition to predominantly Methanosarcinaceae.

4. The method of claim 2, wherein a population of at least one syntrophincreases from a period in which the population of methanogens is predominantly Methanosaeta to a period in which the population of methanogens is predominantly Methanosarcinaceae.

5. The method of claim 4, wherein the at least one syntroph is selected fromthe group consisting of DMER64, Mesotoga, Synergistaceae, Clostridium_sensu_stricto_1,Pelotomaculum, and Syntrophomonas.

6. The method of claim 4, wherein the at least one syntroph is selected fromthe group consisting of Smithella and Syntrophobacher.

7. The method of claim 1, wherein the OLR of the anaerobic digester iscontrolled to be at least 2.5 kg / VS / m3 / d over at least 5 days. Confidential - Company Proprietary8. The method of claim 1, wherein the OLR of the anaerobic digester iscontrolled to be in a range of from 3 to 30 kg / VS / m3 / d over at least 10 days.

9. The method of claim 7, wherein a ratio of solids retention time (SRT) tohydraulic retention time (HRT) of the digester is controlled to be greater than 2.

10. The method of claim 8, wherein a ratio of solids retention time (SRT) tohydraulic retention time (HRT) of the digester is controlled to be in a range of from 3 to 15.

11. The method of claim 1, further comprising condensing at least a portion ofthe evaporate to provide a condensate and recovering ammonia from the condensate.

12. The method of claim 1, wherein the portion of the fluid that has beenbiochemically treated in the anaerobic digester is fed to a vacuum chamber in which thevacuum pressure is applied, and at least a portion of the thickened fluid is recycled to theanaerobic digester where it is further treated.

13. A method for treating a fluid that includes a particulate fraction and asoluble fraction, the method comprising: feeding the fluid to an anaerobic digester; physically and biochemically transforming solids in the particulate fraction of the fluid with microbes in the anaerobic digester; subjecting at least a portion of the fluid that has been biochemically treated in theanaerobic digester to a vacuum pressure to evaporate off at least a portion of the solublefraction of the fluid as evaporate and to provide a thickened fluid, wherein the evaporate includes carbon dioxide and ammonia; further treating the thickened fluid in the anaerobic digester so that it is further physically and biochemically transformed; and condensing at least a portion of the evaporate in the presence of a sufficient amount of water to provide a condensate in which at least some of the ammonia forms ammonium and at least some of the carbon dioxide forms carbonic acid, and causing at least one of ammonium carbonate and ammonium bicarbonate to be formed in the condensate.

14. The method of claim 13, further comprising controlling at least oneparameter, including an organic loading rate (OLR) in the anaerobic digester over a sufficientduration, so that a population of methanogens in the anaerobic digester transitions topredominantly Methanosarcinaceae.

15. The method of claim 13, further comprising adding a source of carbondioxide to the condensate that is separate from the carbon dioxide in the evaporate. Confidential - Company Proprietary16. The method of claim 13, further comprising combining a chlorine sourcewith the condensate and reacting chlorine in the chlorine source with the ammonium in the condensate to produce chloramine.

17. The method of claim 13, wherein no pH adjusting agent is added to theanaerobic digester to increase the pH of the fluid.

18. The method of claim 13, wherein the portion of the fluid that has been biochemically treated in the anaerobic digester is fed to a vacuum chamber in which the vacuum pressure is applied, and at least a portion of the thickened fluid is recycled to the anaerobic digester where it is further treated. Confidential - Company Proprietary

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