Ammonium Inhibition Research Articles

Novel bioaugmentation process to enhance anaerobic digestion of municipal sewage sludge

The imperative shift to a circular economy, driven by population growth, urbanization, and economic development, highlights the importance of recovering resources from waste streams. Anaerobic digestion (AD) is promising for stabilizing organic wastes and producing renewable methane-rich biogas. However, AD faces challenges, including operational instability, fatty acids accumulation, ammonium inhibition, and low yields. This study investigates the applicability of a novel Ydro Process® bioaugmentation on municipal sewage sludge anaerobic digestion. The process led to pre-hydrolysis of sewage sludge, as evidenced by an increase in volatile fatty acids (VFA), from 134 mg HAc/L in raw TWAS to 997 mg HAc/L in augmented sludge and improved soluble chemical oxygen demand (SCOD) yields. Additionally, the viscosity decreased by 41 % in the Ydro Process® bioaugmented sludge. Biogas production significantly increased from 732 mL/day to 1338 mL/day during semi-continuous digestion, with methane yield rising from 116 mL/g TCODadded to 218 mL/g TCODadded. Microbial analysis revealed an enhanced population of hydrogenotrophic methanogens.

Elucidating the role of sub-thermophilic temperature and pre-hydrolyzation for effective upgrading scheme of old swine manure digesters

Solids concentration, temperature, and digester configuration were subjected to biomethanation study to identify effective retrofitting schemes for old swine waste digesters. Batch assays were commenced to determine an appropriate scenario at 30–55 °C and total solids 1–3 %TS. Sub-thermophilic temperature (45 °C) was found desirable with an additional 11.1 % methane yield, while digestion at higher TS induced ammonium inhibition. Subsequent batch experiments lasted 72 hrs for hydrolytic-acidogenic assessment under various temperatures. Heating control at 45 °C and 55 °C for 24 hrs increased hydrolysis efficiency 4.6–5.7 folds above control but showed no significant difference (α = 0.05) between them. Limited heat supply from biogas engine dictated the continuous digestion study to operate pre-hydrolysis reactor at maximum temperature of 45 °C. The two-stage strategy demonstrated best overall performances at the sub-thermphilic combination, raising methane yield by 35.4 %. Next-Generation Sequencing indicated remarkable shifts in abundance and diversity, especially for hydrolytic organisms, which expanded from 54 to 70.2 % by sub-thermophilic temperature.

Functional Redundant Microbiome Enhanced Anaerobic Digestion Efficiency under Ammonium Inhibition Conditions.

Revealing the role of functional redundancy is of great importance considering its key role in maintaining the stability of microbial ecosystems in response to various disturbances. However, experimental evidence on this point is still lacking due to the difficulty in "manipulating" and depicting the degree of redundancy. In this study, manipulative experiments of functional redundancy were conducted by adopting the mixed inoculation strategy to evaluate its role in engineered anaerobic digestion systems under ammonium inhibition conditions. The results indicated that the functional redundancy gradient was successfully constructed and confirmed by evidence from pathway levels. All mixed inoculation groups exhibited higher methane production regardless of the ammonium level, indicating that functional redundancy is crucial in maintaining the system's efficiency. Further analysis of the metagenome-assembled genomes within different functional guilds revealed that the extent of redundancy decreased along the direction of the anaerobic digestion flow, and the role of functional redundancy appeared to be related to the stress level. The study also found that microbial diversity of key functional populations might play a more important role than their abundance on the system's performance under stress. The findings provide direct evidence and highlight the critical role of functional redundancy in enhancing the efficiency and stability of anaerobic digestion.

Effect of Ecuadorian natural zeolite on the performance of anaerobic digestion of swine waste in semicontinuous regime

This study explores the potential of zeolite as an amendment to mitigate ammonium inhibition in the anaerobic digestion of swine waste. Two 50 L reactors, one with and one without zeolite amendment were operated at an OLR of 3.0 g VS L−1d−1 for 130 days, and fed with swine waste from a full-scale pig farm. Under these conditions, zeolite doses of 4 g L−1 allowed total ammonia nitrogen (TAN) concentrations to be kept below 1000 mgNH3-N L−1. The zeolite-amended reactor not only showed an average increase of 8% in methane production under stable conditions but also exhibited 34% reduction in H2S concentrations in the biogas, compared to the reactor without zeolite. The community of archaea originating from the inoculum was conserved in the reactor with zeolite amendment, particularly the acetoclastic methanogens of the genus Methanosaeta. On the other hand, in the reactor without zeolite addition, the microbial community went from being dominated by the acetoclastic methanogen Methanosaeta to having a high relative abundance of hydrogenotrophic methanogens. The zeolite addition also favoured the reactor stability, prevented foaming, and produced an enriched natural zeolite with N, P and K. However, additional studies on the potential of enriched zeolite as a fertilizer are required, which could make the use of zeolite in Anaerobic Digestion of swine waste not only energetically favourable but also economically feasible.

Effect of ammonium/nitrate ratio on microalgae continuous cultures: Species-specificity of nutrient uptake and modelling perspectives

Microalgae-based wastewater treatment is promising in view of circularity as they can uptake most of the nitrogen sources usually present in waste streams: ammonium, nitrite and nitrate as well as some organic species. However, regulation pathways are not fully understood yet. This complexity is a challenge for accurate modelling of these processes, necessary for the design and optimization of wastewater treatment systems. It is generally observed in microalgae that the presence of ammonium inhibits the assimilation of other nitrogen compounds. This was confirmed in this work for Synechocystis sp. PCC6803, even in acclimated continuous cultures. A different uptake regulation was instead observed for the species Auxenochlorella protothecoides, able to uptake nitrate even when high concentrations of ammonium are present. From a modelling perspective, the Droop model was implemented to account for the exploitation of both ammonium and nitrate: a switch function was used to describe the ammonium inhibition, but this was applicable for Synechocystis only. For A. protothecoides, a changing nutrient uptake rate is needed, which was found to be function of the dissolved nitrogen concentration. A bioinformatic analysis was performed to infer putative players involved in nitrogen uptake, accounting for the different behavior of the two taxonomic groups.

Metabolic Regulation of Mesophilic Methanosarcina barkeri to Ammonium Inhibition.

Undesirable ammonium concentrations can lead to unstable anaerobic digestion processes, and Methanosarcina spp. are the representative methanogens under inhibition. However, no known work seems to exist for directly exploring the detailed metabolic regulation of pure cultured representative Methanosarcina spp. to ammonium inhibition. We used transcriptomics and proteomics to profile the metabolic regulation of Methanosarcina barkeri to 1, 4, and 7 g N/L of total ammoniacal nitrogen (TAN), where free ammonia concentrations were between 1.5 and 36.1 mg N/L. At the initial stages of ammonium inhibition, the genes participating in the acquisition and assimilation of reduced nitrogen sources showed significant upregulation where the minimal fold change of gene transcription was about 2. Apart from nitrogen metabolism, the transcription of some genes in methanogenesis also significantly increased at the initial stages. For example, the genes encoding alternative heterodisulfide reductase subunits (HdrAB), energy-converting hydrogenase subunit (EchC), and methanophenazine-dependent hydrogenase subunits (VhtAC) were significantly upregulated by at least 2.05 times. For the element translocation at the initial stages, the genes participating in the uptake of ferrous iron, potassium ion, and molybdate were significantly upregulated with a minimal fold change of 2.10. As the cultivation proceeded, the gene encoding the cell division protein subunit (FtsH) was significantly upregulated by 13.0 times at 7 g N/L of TAN; meanwhile, an increment in OD600 was observed at the terminal sampling point of 7 g N/L of TAN. The present study explored the metabolic regulation of M. barkeri in stress response, protein synthesis, signal transduction, nitrogen metabolism, methanogenesis, and element translocation. The results would contribute to the understanding of the metabolic effects of ammonium inhibition on methanogens and have significant practical implication in inhibited anaerobic digestion.

PDX1.1-dependent biosynthesis of vitamin B6 protects roots from ammonium-induced oxidative stress

Despite serving as a major inorganic nitrogen source for plants, ammonium causes toxicity at elevated concentrations, inhibiting root elongation early on. While previous studies have shown that ammonium-inhibited root development relates to ammonium uptake and formation of reactive oxygen species (ROS) in roots, it remains unclear about the mechanisms underlying the repression of root growth and how plants cope with this inhibitory effect of ammonium. In this study, we demonstrate that ammonium-induced apoplastic acidification co-localizes with Fe precipitation and hydrogen peroxide (H2O2) accumulation along the stele of the elongation and differentiation zone in root tips, indicating Fe-dependent ROS formation. By screening ammonium sensitivity in T-DNA insertion lines of ammonium-responsive genes, we identified PDX1.1, which is upregulated by ammonium in the root stele and whose product catalyzes de novo biosynthesis of vitamin B6. Root growth of pdx1.1 mutants is hypersensitive to ammonium, while chemical complementation or overexpression of PDX1.1 restores root elongation. This salvage strategy requires non-phosphorylated forms of vitamin B6 that are able to quench ROS and rescue root growth from ammonium inhibition. Collectively, these results suggest that PDX1.1-mediated synthesis of non-phosphorylated B6 vitamers acts as a primary strategy to protect roots from ammonium-dependent ROS formation.

Effect of high concentration of ammonium on production of n-caproate: Recovery of a high-value biochemical from food waste via lactate-driven chain elongation.

Ammonium accumulation is inevitable during the fermentation of food waste (FW), challenging the application of chain elongation process upgrading FW into the high-value biochemical n-caproate, which is a medium chain carboxylate. This study is the first to investigate ammonium inhibition of lactate-driven chain elongation process. The short-term exposure of a Clostridium IV-dominated chain elongating reactor microbiome at an ammonium concentration of 1-4g L-1 linearly decreased n-caproate production by 25-80%. High levels of ammonium (≥5g L-1) could cause failure of chain elongation, shifting the product from n-caproate to propionate. The involved mechanisms revealed that ammonium reshaped the microbial community from Clostridium IV domination to Clostridium IV and Propionibacterium co-domination (based on 16S rRNA sequencing) and reduced the activities of key enzymes involved in the reversed β-oxidization pathway. We propose an effective strategy from our study, which is the first one to do in our knowledge, to upgrade raw FW without dilution to n-caproate: lowering the ammonium accumulation to 1.0g L-1 at the setup phase for adaptation and prolonging the hydraulic retention time (10days) during the operation phase for the colonization of chain-elongation bacteria. These findings lay a foundation for the implementation of the LCE process on FW, providing an alternative way to alleviate the global FW crisis.

Photon number based anaerobic digestion process for efficient bio-methane conversion from ammonium-rich feedstock: Performance evaluation and practical potential

In this study, an innovative photon number-based illuminated anaerobic digestion process for bio-methane conversion from ammonium-rich waste has been investigated. The photon flux density and illumination time showed significant effect on ammonium-rich anaerobic digestion. Further, the number of photons, as the integration of two lighting parameters, was found to be a determining factor in illuminated bioprocess for the first time. The effective range of photon number for mitigating ammonium inhibition was observed at 1.16 × 104–1.86 × 104 μmol∙day−1∙L−1. Correspondingly, the result of ATP and coenzyme F420 indicated that illumination with effective number of photons could activate the methanogens responsible for alleviation of ammonia inhibition. In addition, at different ammonium nitrogen concentration, the illuminated bioreactors with effective photon number of 1.25 × 104 μmol∙day−1∙L−1 always showed 2-fold higher methane yield than dark bioreactors. Moreover, the bioreactors with different working volumes under the same photon number condition showed similar methane yield, revealing that the photon number is the key factor for optimizing the illumination condition of anaerobic digestion in treating ammonium-rich substrate. Besides, the illuminated bioreactor with effective photon number condition was stable and well-performed under high ammonium concentration with long-term 89-day semi-continuous operation. This work provides a promising approach to build an easy-operated green recovery system for efficient bio-methane conversion from ammonium-rich waste.

Comparisons of nitrite accumulation, microbial behavior and nitrification kinetic in continuous stirred tank (ST) and plug flow (PF) moving bed biofilm reactors

Two types of continuous stirred tank moving bed biofilm reactors (ST-MBBR) and plug flow MBBR (PF-MBBR) were compared for nitrification. PF-MBBR showed strong shock resistance to temperature, and ammonium oxidation ratio (AOR) was 9.63% higher than that in the ST-MBBR, although the average biomass and biofilm thickness of ST-MBBR were 7.32–18.59%, 9.44–14.06% higher than those in the PF-MBBR. Meanwhile, a lower nitrite accumulation ratio (NAR) was observed (54.88%) in the PF-MBBR than the ST-MBBR (78.92%) due to different operation modes, and the divergence was demonstrated by the microbial quantitative analysis. Nitrification kinetics revealed that the temperature coefficient (θ) in the ST-MBBR (1.068) was much higher than that in the PF-MBBR (1.006–1.015), proving the contrasting nitrification performances caused by temperature shock. According to the Monod equation, the half-saturation coefficient (KN) in the ST-MBBR was 0.19 mg/L while it varied around 0.12–0.24 mg/L in the PF-MBBR, revealing various NH4+ affinity owing to different biofilm thickness and microbial composition. Finally, MBBR optimization related to operation mode, temperature, and free ammonium (FA) inhibition for nitrite accumulation was discussed.

Enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (Km(app)_NH3 ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (Km(app)_NO2- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of “Ca. N. kreftii” indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.

Niche Differentiation of Bacterial Versus Archaeal Soil Nitrifiers Induced by Ammonium Inhibition Along a Management Gradient.

Soil nitrification, mediated mainly by ammonia oxidizing archaea (AOA) and bacteria (AOB), converts ammonium (NH4+) to nitrite (NO2−) and thence nitrate (NO3−). To better understand ecological differences between AOA and AOB, we investigated the nitrification kinetics of AOA and AOB under eight replicated cropped and unmanaged ecosystems (including two fertilized natural systems) along a long-term management intensity gradient in the upper U.S. Midwest. For five of eight ecosystems, AOB but not AOA exhibited Haldane kinetics (inhibited by high NH4+ additions), especially in perennial and successional systems. In contrast, AOA predominantly exhibited Michaelis-Menten kinetics, suggesting greater resistance to high nitrogen inputs than AOB. These responses suggest the potential for NH4+-induced niche differentiation between AOA and AOB. Additionally, long-term fertilization significantly enhanced maximum nitrification rates (Vmax) in the early successional systems for both AOA and AOB, but not in the deciduous forest systems. This was likely due to pH suppression of nitrification in the acidic forest soils, corroborated by a positive correlation of Vmax with soil pH but not with amoA gene abundance. Results also demonstrated that soil nitrification potentials were relatively stable, as there were no seasonal differences. Overall, results suggest that (1) NH4+ inhibition of AOB but not AOA could be another factor contributing to niche differentiation between AOA and AOB in soil, and (2) nitrification by both AOA and AOB can be significantly promoted by long-term nitrogen inputs.

A Novel Nitrogen Removal Technology Pre-Treating Chicken Manure, Prior to Anaerobic Digestion

Chicken manure is an agricultural by-product that is a problematic feedstock for anaerobic digestion due to its high nitrogen content inhibiting methane yields. This research examines a novel pilot-scale method of ammonia stripping, the nitrogen recovery process (NRP) developed by Alchemy Utilities Ltd. The NRP was designed to remove and recover nitrogen from chicken manure and two different operating conditions were examined. Both operating conditions demonstrated successful nitrogen removal and recovery. The biochemical methane potential assays were used to compare the digestibility of the NRP-treated chicken manures to that of a fresh chicken manure control. Overall, the biochemical methane potential assays demonstrated that some NRP-treated chicken manure treatments produced significantly more methane compared to untreated manure, with no inhibition occurring in relation to ammonium. However, some of the NRP-treated chicken manures produced similar or lower methane yields compared to fresh chicken manure. The NRP requires further development to improve the efficiency of the pilot-scale unit for commercial-scale operation and longer-term continuous anaerobic digestion trials are required to determine longer-term methane yield and ammonium inhibition effects. However, these initial results clearly demonstrate the technology’s potential and novel application for decentralised, on-farm nitrogen recovery and subsequent anaerobic digestion of chicken manure.

Improving anaerobic digestion of piggery wastewater by alleviating stress of ammonia using biochar derived from rice straw

Abstract In this study, impact of a rice straw-derived biochar on the anaerobic digestion of piggery wastewater was investigated under different ammonium “stress levels”. The results showed that the biogas yield was decreased from 1293.27mL to 402.31 mL and the COD removal rate was decreased from 70.68% to 38.13% with the NH 4 + -N concentration increasing from 900mg/L to 3500 mg/L. At no ammonium “stress level”, the COD removal rate was increased from 70.68% to 83.75%, and biogas yield was increased from 1293.27mL to 2306mL with the increase of biochar dosage from 0g to 15g. While with the addition of biochar increasing from 0g to 15g, the COD removal rate was increased from 38.13% to 70.38% and the biogas yield was increased from 382 mL to 1878 mL at high ammonium “stress level”. It indicated that the presence of biochar not only could promote biogas yield but also could release the ammonium inhibition.

Effects of encapsulation on the chemical inhibition of anaerobic hydrogen- and methane-producing microbial cells

Encapsulating microbial cells in an alginate matrix enables high-rate anaerobic biological treatment and resource recovery by increasing cell concentration and retention time. To determine whether encapsulation protected microbial cells from inhibitors, copper(II), chromium(VI) (as dichromate), ammonium, and chloroform were amended to hydrogen-producing anaerobic microbial cells that were either suspended or encapsulated in a composite-coated alginate matrix. Encapsulation mitigated dichromate inhibition by 36% based on the dose-response curve slope but increased ammonium inhibition by 210% and had no effect on inhibition by chloroform. This was explained by effective concentrations in the beads after measuring partition coefficients. Encapsulation rendered copper(II) unavailable through apparent chelation because it protected both the hydrogen-producing and a methane-producing community from copper(II) despite its high effective concentration in encapsulation. Understanding the effects that the encapsulation of microbial cells has on chemical inhibition can help expand the application conditions of wastewater treatment or resource recovery and avoid unanticipated toxicity.

Optimization of algae production on urine

Urine is a potential source of nutrients to grow microalgal biomass to be re-used as fertilizer and soil conditioner. In this study the impact of photobioreactor dilution rate on microalgae productivity and photosynthetic efficiency was assessed and used to determine operating conditions to reach both full nitrogen removal from urine and high biomass productivity. In addition, the possibility to work under day/night cycling was tested. To this end, the microalga Chlorella sorokiniana was grown on artificial urine and real human urine in bench-scale panel photobioreactors with short optical paths.At a light intensity of 1530 μmol⋅m−2⋅s-1 photobioreactor productivity and photosynthetic efficiency was demonstrated to be maximal at reactor dilution rates between 0.10 and 0.15h-1. A biomass yield of 1g dry matter per mol of PAR photons was achieved. Biomass concentration, and accordingly nutrient removal efficiency, decreased at increasing reactor dilution rate. The experimental results could be reproduced by model simulations. These simulations allowed to demonstrate that the system must be operated at a dilution rate of less than 0.01 h-1 in order to reach complete nitrogen removal. In that scenario more than half of the potential biomass productivity is lost due to severe self-shading within the algal culture. Experiments with real human urine illustrated the problem of incomplete nitrogen removal and ammonium inhibition of growth at too high dilution rates. It is therefore suggested to apply an optimized pre-dilution of pure urine prior to treatment in a photobioreactor.Experiments under day-night cycles demonstrated that microalgal cultures quickly acclimate to such variable light conditions. Additional model simulations illustrated that a phototrophic system is most effective when diluted urine is fed to the photobioreactor during day time only. In that situation the lowest nitrogen concentration in the effluent can be reached at a maximal areal removal rate and photosynthetic efficiency.

Metabolic performance of anaerobic digestion of chicken manure under wet, high solid, and dry conditions

The anaerobic digestion (AD) of chicken manure as a solo substrate has been challenging due to the ammonium inhibition effects when adopting a high organic loading rate (OLR). In this study, through increasing both the total solid in the feeding materials from 5% to 20%, and the OLR from 1.7 to 7.1 g-volatile solids (VS)/(L·d), the AD of chicken manure under wet, high solid, and dry conditions, with a fixed hydraulic retention time of 20 days, was investigated. The results obtained indicated that the wet AD system could achieve a methane yield of 0.28 L/g-VS and a low volatile fatty acid level. However, the process deteriorated under dry conditions, and methane formed mainly through acetate oxidation and methanogenesis. Methanosarcina and Methanoplasma were found to be more tolerant But, whether the dry AD of chicken manure can survive an ammonia-stressed environment when the OLR is lowered, still needs investigation.

Cow manure as additive to a DMBR for stable and high-rate digestion of food waste: Performance and microbial community

Cow manure (CM) was added to a dynamic membrane bioreactor (DMBR) operated under anaerobic condition for enhancing food waste (FW) digestion for over 300 days with stepwise increase of organic loading rates (OLRs) from 1.07 to 11.9 g COD/L/day. At a FW/CM ratio of 3.5:1 (based on volatile solids), the mixed liquor pH was always above 8.0 and no apparent volatile fatty acids (VFAs) accumulation occurred even at the highest OLR of 11.9 g COD/L/day (hydraulic retention time as 10 days and solid retention time as 15.5 days, correspondingly), indicating a very stable operation condition which resulted in an average CH4 yield as high as 250 mL/g COD and CH4 production as high as 2.71 L CH4/L/day. The hardly biodegradable organic components, such as cellulose, hemicellulose, and lignin, were effectively degraded by 78.3%, 58.8%, and 47.5%, respectively. Significantly high anaerobic digestion reaction ratios, especially the hydrolysis ratio which is usually the limiting factor, were calculated based on experimental results. Furthermore, the high lignocellulase contents and coenzyme F420 levels, along with the decrease of cellulose crystallinity from 72.6% to 16.4% in the feedstock, provided strong evidence of an enhanced biological activity by CM addition. By high-throughput sequencing analysis, more abundant and diverse bacterial, archaeal, and fungal genera were identified from the DMBR sludge. With CM addition, the biodegradation of lignocellulose might have produced sufficient H2 and CO2 for the hydrogenotrophic methanogens such as Methanoculleus, Methanomassiliicoccus, and Methanobacterium, which were highly tolerant to ammonium inhibition, and then the elevated ammonium level would have provided high buffering capacity in the DMBR thus ensuring a stable condition for high rate FW digestion and CH4 production.

Recovery behavior of high ammonium inhibition in a sequencing batch biofilm reactor

Few studies have investigated the process of microbial recovery in wastewater inhibited by high-strength ammonium but focused on the inhibition effects only. In this study, one contrast way and four efficient ways were developed and applied to five parallel sequencing batch biofilm reactors (SBBRs). The effluent ammonia nitrogen (NH4+-N), nitrite nitrogen (NO2−-N), nitrate nitrogen (NO3−-N), total nitrogen (TN), chemical oxygen demand (COD) and total phosphorus (TP) concentrations were investigated to assess the recovery effects of removal rates as well as the recovery mechanism. Results showed that all of these methods have a positive effect, among which introducing algae was optimum for the fast removal recovery of TN and TP within 4 days. And its recovery time is at least 8 days shorter than that in natural recovery. The recovery ways proposed in this study can be potentially applied in the emergency treatment in SBBR system.

Ammonium inhibition of the intercalation of dicarboxylic acid molecules into octacalcium phosphate layer by substitution

Octacalcium phosphate (OCP) is an attractive material for becoming the centerpiece of medical combination products as a layered calcium phosphate because it exhibits excellent biocompatibility and because various molecules can be intercalated between its layers. However, the factors that govern various events, such as molecular substitution into an OCP unit lattice, remain unclear. Herein, we focused on monovalent cations, including NH4+ and Na+, which are commonly used as agents to adjust the pH and ionic strength. This study aims to demonstrate the manner in which NH4+ inhibits the intercalation of thiomalate (SH–malate), which is an antirheumatic drug precursor, into the OCP unit lattice during SH–malate-substituted OCP (OCP–SH–malate) formation. With increasing NH4+ concentration, the amount of SH–malate substituted into the OCP unit lattice decreased without a change in the crystal morphology. This characteristic is undesirable for preparing OCP combination products because NH4+, which is commonly used in the buffer solutions, will strongly inhibit the intercalation of drug molecules into OCP.