Ammonia-oxidizing Microorganisms Research Articles

The Stochastic Assembly of Nitrobacter winogradskyi-Selected Microbiomes with Heterotrophs from Sewage Sludge or Grassland Soil.

Chemolitho-autotrophic microorganisms like the nitrite-oxidizing Nitrobacter winogradskyi create an environment for heterotrophic microorganisms that profit from the production of organic compounds. It was hypothesized that the assembly of a community of heterotrophic microorganisms around N. winogradskyi depends on the ecosystem from which the heterotrophs are picked. To test this hypothesis, pure cultures of N. winogradskyi were grown in continuously nitrite-fed bioreactors in a mineral medium free of added organic carbon that had been inoculated with diluted sewage sludge or with a suspension from a grassland soil. Samples for chemical and 16S rRNA gene amplicon analyses were taken after each volume change in the bioreactor. At the end of the enrichment runs, samples for shotgun metagenomics were also collected. Already after two volume changes, the transformations in community structure became less dynamic. The enrichment of heterotrophs from both sewage and soil was highly stochastic and yielded different dominant genera in most of the enrichment runs that were independent of the origin of the inoculum. Hence, the hypothesis had to be refuted. Notwithstanding the large variation in taxonomic community structure among the enrichments, the functional compositions of the communities were statistically not different between soil- and sludge-based enrichments. IMPORTANCE In the process of aerobic nitrification, nitrite-oxidizing bacteria together with ammonia-oxidizing microorganisms convert mineral nitrogen from its most reduced appearance, i.e., ammonium, into its most oxidized form, i.e., nitrate. Because the form of mineral nitrogen has large environmental implications, nitrite-oxidizing bacteria such as Nitrobacter winogradskyi play a central role in the global biogeochemical nitrogen cycle. In addition to this central role, the autotrophic nitrite-oxidizing bacteria also play a fundamental role in the global carbon cycle. They form the basis of heterotrophic food webs, in which the assimilated carbon is recycled. Little is known about the heterotrophic microorganisms that participate in these food webs, let alone their assembly in different ecosystems. This study showed that the assembly of microbial food webs by N. winogradskyi was a highly stochastic process and independent of the origin of the heterotrophic microorganisms, but the functional characteristics of the different food webs were similar.

The Effects of Quinone Imine, a New Potent Nitrification Inhibitor, Dicyandiamide, and Nitrapyrin on Target and Off-Target Soil Microbiota.

ABSTRACTDicyandiamide (DCD) and nitrapyrin (NP) are nitrification inhibitors (NIs) used in agriculture for over 40 years. Recently, ethoxyquin (EQ) was proposed as a novel potential NI, acting through its derivative quinone imine (QI). Still, the specific activity of these NIs on the different groups of ammonia-oxidizing microorganisms (AOM), and mostly their effects on other soil microbiota remain unknown. We determined the impact of QI, and comparatively of DCD and NP, applied at two doses (regular versus high), on the function, diversity, and dynamics of target (AOM), functionally associated (nitrite-oxidizing bacteria-NOB), and off-target prokaryotic and fungal communities in two soils mainly differing in pH (5.4 versus 7.9). QI was equally effective to DCD but more effective than NP in inhibiting nitrification in the acidic soil, while in the alkaline soil QI was less efficient than DCD and NP. This was attributed to the higher activity of QI toward AOA prevailing in the acidic soil. All NIs induced significant effects on the composition of the AOB community in both soils, unlike AOA, which were less responsive. Beyond on-target effects, we noted an inhibitory effect of all NIs on the abundance of NOB in the alkaline soil, with Nitrobacter being more sensitive than Nitrospira. QI, unlike the other NIs, induced significant changes in the composition of the bacterial and fungal communities in both soils. Our findings have serious implications for the efficiency and future use of NIs on agriculture and provide unprecedented evidence for the potential off-target effects of NIs on soil microbiota.IMPORTANCE NIs could improve N use efficiency and decelerate N cycling. Still, we know little about their activity on the distinct AOM groups and about their effects on off-target soil microorganisms. Here, we studied the behavior of a new potent NI, QI, compared to established NIs. We show that (i) the variable efficacy of NIs across soils with different pH reflects differences in the inherent specific activity of the NIs to AOA and AOB; (ii) beyond AOM, NIs exhibit negative effects on other nitrifiers, like NOB; (iii) QI was the sole NI that significantly affected prokaryotic and fungal diversity. Our findings (i) highlight the need for novel NI strategies that consider the variable sensitivity of AOM groups to the different NIs (ii) identify QI as a potent AOA inhibitor, and (iii) stress the need for monitoring NIs’ impact on off-target soil microorganisms to ensure sustainable N fertilizers use and soil ecosystem functioning.

Changes in the Species and Functional Composition of Activated Sludge Communities Revealed Mechanisms of Partial Nitrification Established by Ultrasonication

To achieve energy-efficient shortcut nitrogen removal of wastewater in the future, selective elimination of nitrite-oxidizing bacteria (NOB) while enriching ammonia-oxidizing microorganisms is a crucial step. However, the underlying mechanisms of partial nitrification are still not well understood, especially the newly discovered ultrasound-based partial nitrification. To elucidate this issue, in this study two bioreactors were set up, with one established partial nitrification by ultrasonication while the other didn't. During the operation of both reactors, the taxonomic and functional composition of the microbial community were investigated through metagenomics analysis. The result showed that during ultrasonic partial nitrification, ammonia-oxidizing archaea (AOA), Nitrososphaerales, was enriched more than ammonia-oxidizing bacteria (AOB), Nitrosomonas. The enrichment of microorganisms in the community increased the abundance of genes involved in microbial energy generation from lipid and carbohydrates. On the other hand, the abundance of NOB, Nitrospira and Nitrolancea, and Comammox Nitrospira decreased. Selective inhibition of NOB was highly correlated with genes involved in signal transduction enzymes, such as encoding histidine kinase and serine/threonine kinase. These findings provided deep insight into partial nitrification and contributed to the development of shortcut nitrification in wastewater treatment plants.

The Responses of Ammonia-Oxidizing Microorganisms to Different Environmental Factors Determine Their Elevational Distribution and Assembly Patterns.

The assembly mechanisms shaping the elevational patterns of diversity and community structure in ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) are not well understood. We investigated the diversities, co-occurrence network patterns, key drivers, and potential activities of AOA and AOB communities along a large altitudinal gradient. The α-diversity of the AOA communities exhibited a monotonically decreasing pattern with increasing elevation, whereas a sinusoidal pattern was observed for the AOB communities. The mean annual temperature was the single factor that most strongly influenced the α-diversity of the AOA communities; however, the interactions of plant richness, soil conductivity, and total nitrogen made comparable contributions to the α-diversity of the AOB communities. Moreover, the β-diversities of the AOA and AOB communities were divided into two distinct clusters by elevation, i.e., low- (1800-2600m) and high-altitude (2800-4100m) sections. These patterns were attributed mainly to the soil pH, followed by variations in plant richness along the altitudinal gradient. In addition, the AOB communities were more important to the soil nitrification potential in the low-altitude section, whereas the AOA communities contributed more to the soil nitrification potential in the high-altitude section. Overall, this study revealed the key factors shaping the elevational patterns of ammonia-oxidizing communities and might predict the consequences of changes in ammonia-oxidizing communities.

Diversity distribution and characteristics of comammox in different ecosystems

The discovery of complete ammonia oxidizers (comammox), which can oxidize ammonia into nitrate, has recently changed the concept of traditional nitrification. However, comparative studies on the analysis of comammox microbial community in different ecosystems are still scarce. In this study, the distribution and diversity of the comammox microbial community in farmlands, riparian zones, and river sediments in summer and winter were investigated by high-throughput sequencing. And the relative abundance of ammonia-oxidizing microorganisms was measured via their amoA genes of real-time quantitative polymerase chain reaction (qPCR). The relationships between ammonia oxidation microorganisms and the environmental factors were further analyzed. The abundance of comammox clade A was one order of magnitude lower than that of ammonia-oxidizing archaea (AOA) but higher than that of ammonia-oxidizing bacteria (AOB). The abundance of comammox was higher in summer than in winter and higher in farmland soils (1.81 ± 0.95 × 107 copies g−1) than in riparian zones and river sediments. Meanwhile, Candidatus Nitrospira nitrosa were the most widespread comammox in most samples (up to 86.31%), followed by Candidatus Nitrospira nitrificans, with a low abundance of Candidatus Nitrospira inopinata (lower than 0.61%). Furthermore, the abundance of comammox clade A had a significantly negative correlation with pH and NH4+ concentration (P < 0.05). The study revealed the potential advantages of comammox in farmlands and may be conducive to further research on comammox in microbial nitrogen cycling.

Effects of Ionized Water Addition on Soil Nitrification Activity and Nitrifier Community Structure

Water ionization is an efficient physical water treatment technology, and crop water and nutrient use efficiencies can be improved using ionized water for irrigation. In order to explore the effect of ionized water on soil nitrification and nitrifying microorganisms, we conducted a laboratory soil incubation experiment with the addition of ionized water and ordinary water under different soil water contents (equal to 30%, 60%, 100% and 175% of the field capacity, θFC). During the soil incubation, we analyzed soil inorganic nitrogen transformation, ammonia oxidation gene abundances and nitrifying microbial community structure. The results showed that, no matter adding ordinary water or ionized water, the soil nitrification rate and the abundance of ammonia oxidizing bacteria in the 100%θFC treatment were significantly higher than those in other water conditions, while the abundance of ammonia oxidizing archaea was not affected by the soil water content. With the same soil water content, the nitrification rate of ionized water treatment was stronger than that of the ordinary water treatment. Although the absolute abundance of ammonia-oxidizing microorganisms in ionized water treatment was significantly lower than that of ordinary water (p &lt; 0.05), the relative abundance of some dominant nitrifying microbial genera in the ionized water treatment was significantly higher (p &lt; 0.05). The dominant genera may play a key role in the nitrification process. The results show that ionized water irrigation can significantly promote the nitrification of silt loam soil, especially under 100%θFC conditions, and may regulate soil nitrification by affecting some dominant nitrifying microorganisms. This study provides a theoretical basis for understanding the biological regulation mechanism of ionized water irrigation on soil nutrient transformation and for application of ionized water to field irrigation.

Responses of soil ammonia-oxidizing microorganisms to simulated nitrogen deposition in a natural Castanopsis carlesii forest

Subtropical region of China is one of the global hotspots receiving nitrogen deposition. Nitrogen deposition could affect the abundance and community structure of ammonia oxidizers including ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and complete ammonia oxidizer (comammox Nitrospira), with consequences on soil nutrient cycling that are driven by microorganisms. There is limited understanding for the newly discovered comammox Nitrospira in the subtropical forest soils. Here, we investigated the effect of simulated N deposition on abundances of soil ammonia oxidizers in the Castanopsis fargesii Nature Reserve in Xinkou Town, Sanming City, Fujian Province, China. Soil samples were collected from the field plots which received long-term nitrogen deposition with different dosages, including: CK, no additional treatment; LN, low nitrogen deposition treatment, dosage of 40 kg N·hm-2·a-1; and HN, high nitrogen deposition treatment, dosage of 80 kg N·hm-2·a-1. After 8-year treatment, simulated N deposition decreased soil pH and organic matter content, and increased nitrate content. We failed to amplify the amoA gene of AOB in the tested soils. High nitrogen deposition increased the abundance of AOA, but did not affect the abundance of comammox Nitrospira clade A and clade B. The ratio of comammox Nitrospira to AOA decreased with N addition, indicating that N addition weakened the role of comammox Nitrospira in nitrification in the subtropical forest soils. However, there were strong non-specific amplifications for both comammox Nitrospira clades A and B, highlighting the demand for the development of high coverage and specificity primers for comammox Nitrospira investigations in the future. The abundance of comammox Nitrospira clade A was positively correlated with total nitrogen (TN) and NH4+ concentration, while that of clade B was positively associated with soil organic carbon (SOC), TN and NH4+ Concentration. Overall, our findings demonstrated that simulated N deposition increased the relative importance of AOA in nitrification in the natural Castanopsis carlesii forest soil. These findings could provide theoretical support in coping with global change and N deposition in these regions.

Comammox bacteria predominate among ammonia-oxidizing microorganisms in municipal but not in refinery wastewater treatment plants

Comammox bacteria have proved to be one dominant and significant ammonia-oxidizing microorganisms (AOMs) in municipal wastewater treatment plants (WWTPs), however, it still remains unknown about their abundance and diversity in industrial WWTPs. In this study, activated sludge samples from 8 municipal WWTPs and 6 industrial WWTPs treating refinery wastewater were taken and analyzed using qPCR and amoA gene sequencing. Intriguingly, quantitative real-time PCR (qPCR) results suggested that comammox bacteria had a higher numerical abundance compared with ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in municipal WWTPs but did not in refinery WWTPs. Moreover, comammox amoA sequences obtained from high-throughput sequencing were retrieved from all the 8 municipal samples but only 1 industrial sample. Further phylogenetic analysis revealed that N. nitrosa cluster accounted for as high as 79.56% of the total comammox affiliated sequences, which was the most numerically abundant comammox species in municipal WWTPs. This study provided new insights into the abundance and diversity of comammox bacteria in the biological nitrification process in municipal and refinery wastewater treatment systems.

Effects of Soil Salinity on Nitrification and Ammonia-Oxidizing Microorganisms in Coastal Reclaimed Farmland Soil

Effects of Soil Salinity on Nitrification and Ammonia-Oxidizing Microorganisms in Coastal Reclaimed Farmland Soil

Spatiotemporal dynamics of ammonia-oxidizing archaea and bacteria contributing to nitrification in sediments from Bohai Sea and South Yellow Sea, China

Nitrification is a central process in nitrogen cycle in the ocean. Ammonia-oxidizing archaea (AOA) and bacteria (AOB) play significant roles in ammonia oxidation which is the first and rate-limiting step in nitrification, and their differential contribution to nitrification is an important issue, attracting extensive attention. In this study, based on the quantification of archaeal and bacterial amoA gene and the measurement of potential nitrification rate (PNR), we investigated the spatiotemporal dynamics of PNRs and the amoA gene abundance and transcript abundance of aerobic ammonia oxidizers in surface sediments collected in summer and spring across ~900 km of the Bohai Sea and Yellow Sea in China. The results revealed that the contribution of AOA to nitrification was greater than that of AOB in coastal sediments, probably due to salinity and ammonia concentration. Besides, seasons had significant effect on amoA gene abundance and transcript abundance, especially for AOA, while both seasons and sea areas had significant influence on PNR of AOA and AOB. Further analysis showed complex relationships among amoA gene abundances, transcript abundances and PNRs. More importantly, both spatial (geographic distance) and environmental factors were vital in explaining the variations of ammonia-oxidizing microorganism abundances and the PNRs.

Biochar Addition Inhibits Nitrification by Shifting Community Structure of Ammonia-Oxidizing Microorganisms in Salt-Affected Irrigation-Silting Soil.

Biochar has been widely recognized as an effective and eco-friendly ameliorant for saline soils, but information about the mechanism of how biochar influences nitrification in salt-affected agroecosystem remains fragmented. An incubation experiment was performed on the salt-affected soil collected from a three-consecutive-year experiment at biochar application gradients of 7.5 t⋅ha−1, 15 t⋅ha−1 and 30⋅t ha−1 and under nitrogen (N) fertilization. Responses of the nitrification rate (NR), numbers of ammonia monooxygenase (amoA) gene copies, and community structures of ammonia-oxidizing bacteria (AOB) and archaea (AOA) to biochar application were investigated. The results indicated that, under N fertilization, the NR and numbers of amoA-AOB and amoA-AOA gene copies negatively responded to biochar addition. Biochar application increased the community diversity of AOB but decreased that of AOA. Biochar addition and N fertilization shifted the AOB community from Nitrosospira-dominated to Nitrosospira and Nitrosomonas-dominated, and altered the AOA community from Nitrososphaera-dominated to Nitrososphaera and Nitrosopumilus-dominated. The relative abundance of Nitrosospira, Nitrosomonas and Nitrosopumilus decreased, and that of Nitrosovibrio and Nitrososphaera increased with biochar application rate. Soil SOC, pH and NO3−-N explained 87.1% of the variation in the AOB community, and 78.1% of the variation in the AOA community was explanatory by soil pH and SOC. The SOC and NO3−-N influenced NR through Nitrosovibrio, Nitrosomonas, Norank_c_environmental_samples_p_Crenarchaeota and amoA-AOB and amoA-AOA gene abundance. Therefore, biochar addition inhibited nitrification in salt-affected irrigation-silting soil by shifting the community structures of AOB and AOA and reducing the relative abundance of dominant functional ammonia-oxidizers, such as Nitrosospira, Nitrosomonas and Nitrosopumilus.

Distribution characteristics of ammonia-oxidizing microorganisms and their responses to external nitrogen and carbon in sediments of a freshwater reservoir, China

Distribution characteristics of ammonia-oxidizing microorganisms and their responses to external nitrogen and carbon in sediments of a freshwater reservoir, China

Substrate loading rates conducive to nitritation in entrapped cell reactors: performance and microbial community structure.

This study aimed to elucidate the boundaries of ammonia and organic loading rates that allow for nitritation in continuous flow phosphorylated-polyvinyl alcohol entrapped cell reactors and to clarify the community structure of microorganisms involving nitrogen transformation in the gel bead matrices. At operating bulk dissolved oxygen concentration of 2mg/L, nitritation was accomplished when the total ammonia nitrogen (TAN) loading rate was ≥ 0.3 kgN/m3/d. At TAN loading rates of ≤ 0.2 kgN/m3 /d, complete oxidation of ammonia to nitrate took place. Nitritation performance dropped with increased chemical oxygen demand (COD) loading rates indicating limitation of nitritation reactor operation at some COD loading conditions. 16S rRNA gene amplicon sequencing revealed that the uncultured Cytophagaceae bacterium, Arenimonas, Truepera, Nitrosomonas, Comamonas, unclassified Soil Crenarchaeotic Group, and uncultured Chitinophagaceae bacterium were highly abundant taxa in the reactors' gel bead matrices. qPCR with specific primers targeting amoA genes demonstrated the coexistence of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea, and Comammox in the gel bead matrices. AOB was likely the main functioning ammonia-oxidizing microorganisms due to the amoA gene being of highest abundance in most of the studied conditions. Nitrite-oxidizing microorganisms presented in less relative abundance than ammonia-oxidizing microorganisms, with Nitrobacter rather than Nitrospira dominating in the group. Results obtained from this study are expected to further the application of nitritation entrapped cell reactors to real wastewater treatment processes.

Metabolic Relationships and Niche Differentiation between Ammonia-Oxidizing Microorganisms and Complete Ammonia Oxidizers in Soil Ecosystems

Metabolic Relationships and Niche Differentiation between Ammonia-Oxidizing Microorganisms and Complete Ammonia Oxidizers in Soil Ecosystems

Responses of Ammonia Oxidizing Microorganisms to Single and Combined Sulfamethazine and Cadmium Exposure in Soil

Responses of Ammonia Oxidizing Microorganisms to Single and Combined Sulfamethazine and Cadmium Exposure in Soil

Accelerated Nitrification Rate and Altered Community Structure of Ammonia-Oxidizing Microorganisms in the Saline-Alkali Tolerant Rice Rhizosphere of Coastal Solonchaks

Accelerated Nitrification Rate and Altered Community Structure of Ammonia-Oxidizing Microorganisms in the Saline-Alkali Tolerant Rice Rhizosphere of Coastal Solonchaks

Cometabolic biodegradation of antibiotics by ammonia oxidizing microorganisms during wastewater treatment processes

Studies on antibiotic removal during wastewater treatment processes are crucial since their release into the environment could bring potential threats to human health and ecosystem. Cometabolic biodegradation of antibiotics by ammonia oxidizing microorganisms (AOMs) has received special attentions due to the enhanced removal of antibiotics during nitrification processes. However, the interactions between antibiotics and AOMs are less well-elucidated. In this review, the recent research proceedings on cometabolic biodegradation of antibiotics by AOMs were summarized. Ammonia oxidizing bacteria (AOB), ammonia oxidizing archaea (AOA) and complete ammonia oxidizers (comammox) played significant roles in both nitrification and cometabolic biodegradation of antibiotics. Antibiotics at varying concentrations might pose inhibiting or stimulating effect on AOMs, influencing the microbial activity, community abundance and ammonia monooxygenase subunit A gene expression level. AOMs-induced cometabolic biodegradation products were analyzed as well as the corresponding pathways for each type of antibiotics. The effects of ammonium availability, initial antibiotic concentration, sludge retention time and temperature were assessed on the cometabolic biodegradation efficiencies of antibiotics. This work might provide further insights into the fate and removal of antibiotics during nitrification processes.

Combined application of biochar and N fertilizer shifted nitrification rate and amoA gene abundance of ammonia-oxidizing microorganisms in salt-affected anthropogenic-alluvial soil

Biochar has been globally recognized as a novel and eco-friendly soil ameliorant to mitigate hazardous effects of salinization on soil health, and mediate carbon and nitrogen (N) nutrient cycling in agricultural ecosystems. However, knowledge pertaining to the regulatory mechanism of biochar application on nitrification in the salt-affected soil is limited. In this study, field plot experiment for three consecutive years and an aerobic incubation experiment with three biochar application rates (7.5, 15, and 30 ton ha−1) and N fertilizer (225 kg N ha−1 yr−1) were conducted. We investigated the effect of combined application of biochar and N fertilizer on soil chemical and microbial properties, nitrification kinetic parameters, and the abundance of ammonia monooxygenase (subunit A, amoA) gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Our results showed that biochar addition increased soil pH, cation exchange capacity, available potassium, and microbial biomass carbon, and decreased the numbers of amoA-AOB gene in the salt-affected soil applied with N fertilizer. The nitrification process and fitted kinetic parameters were negatively responsive to the combined application of biochar and N fertilizer, which reduced the nitrification potential and potential nitrification rate, and prolonged the duration of nitrification. The nitrification rate and abundance of amoA-AOB and amoA-AOA genes decreased under biochar and N fertilizer addition, and biochar played the predominant role in the inhibiting effect, which aggravated with the increase of biochar rate. The AOB dominated the autotrophic nitrification with the nitrification rate having higher positive correlation with abundance of amoA-AOB gene than with that of amoA-AOA gene. The abundance of both amoA-AOB and amoA-AOA genes showed significantly negative correlation with nitrate nitrogen (NO3−-N) concentration. In conclusion, biochar and N fertilizer addition inhibited the autotrophic nitrification rate mainly by greatly reducing the abundance of amoA-AOB gene, which was adaptive to the changing soil microhabitat traits induced by biochar application and N fertilization.

Grazing weakens competitive interactions between active methanotrophs and nitrifiers modulating greenhouse-gas emissions in grassland soils

Grassland soils serve as a biological sink and source of the potent greenhouse gases (GHG) methane (CH4) and nitrous oxide (N2O). The underlying mechanisms responsible for those GHG emissions, specifically, the relationships between methane- and ammonia-oxidizing microorganisms in grazed grassland soils are still poorly understood. Here, we characterized the effects of grazing on in situ GHG emissions and elucidated the putative relations between the active microbes involving in methane oxidation and nitrification activity in grassland soils. Grazing significantly decreases CH4 uptake while it increases N2O emissions basing on 14-month in situ measurement. DNA-based stable isotope probing (SIP) incubation experiment shows that grazing decreases both methane oxidation and nitrification processes and decreases the diversity of active methanotrophs and nitrifiers, and subsequently weakens the putative competition between active methanotrophs and nitrifiers in grassland soils. These results constitute a major advance in our understanding of putative relationships between methane- and ammonia-oxidizing microorganisms and subsequent effects on nitrification and methane oxidation, which contribute to a better prediction and modeling of future balance of GHG emissions and active microbial communities in grazed grassland ecosystems.

Triclocarban shifted the microbial communities and promoted the spread of antibiotic resistance genes in nitrifying granular sludge system

Triclocarban (TCC) is in great market demand especially after the outbreak of COVID-19 pandemic, becoming an emerging pollutant. However, the impacts of TCC on the performance of nitrifying granular sludge system and the occurrence of antibiotic resistance genes (ARGs) were still unknown. This work explored the impacts of different concentrations of TCC on nitrifying granular sludge. Results showed that TCC suppressed the activities of ammonia-oxidizing microorganisms and decreased the abundance of Nitrospira. Adsorption was the main way for the removal of TCC and the biodegradation efficiency of TCC increased to 28.00% under 19.70 mg/L TCC addition. TCC enriched the ARGs and promoted the risks of their transferring in microorganisms. Pseudomonas might not only have strong resistance to TCC, but also propagate ARGs. The removal process of TCC and bacterial communities were important factors to promote the spread of ARGs. Thus, the existence of TCC presented a great environmental risk.