Autotrophic Ammonia-oxidizing Bacteria Research Articles

Feast/famine ratio regulates the succession of heterotrophic nitrification-aerobic denitrification and autotrophic ammonia oxidizing bacteria in halophilic aerobic granular sludge treating saline wastewater

Heterotrophic nitrification-aerobic denitrification (HN-AD) shows innovation potential of wastewater treatment process in a single tank. However, how to enrich HN-AD bacteria in activated sludge to enhance their contribution remained unknown. This study explored the impact of the feast/famine (F/F) ratio on the succession of autotrophic ammonia oxidizing bacteria (AOB) and HN-AD bacteria in a halophilic aerobic granular sludge (HAGS) system. As the F/F ratio decreased from 1/9 to 1/15, the total inorganic nitrogen (TIN) removal performance significantly decreased. The proportion of heterotrophic bacteria was dropped from 79.0 % to 33 %. Accordingly, the relative abundance of Paracoccus decreased from 70.8 % to 25.4 %, and the copy number of the napA gene was reduced from 2.2 × 1010 copies/g HAGS to 8.1 × 109 copies/g HAGS. It found the F/F ratio regulated the population succession of autotrophic AOB and HN-AD bacteria, thereby providing a solution to achieve the enrichment of HN-AD bacteria in HAGS.

Comammox dominate soil nitrification under different N fertilization regimes in semi-arid areas of Northeast China

The newly identified complete ammonia oxidation (Comammox), which is capable of oxidizing ammonia directly to nitrate, has complemented the knowledge of nitrification in the global nitrogen (N) cycle. Knowledge of the community compositions and contributions to nitrification of autotrophic ammonia-oxidizing archaea (AOA) and bacteria (AOB) and Comammox remain void. In this study, the abundance, community compositions, and contributions to nitrification of AOA, AOB, and Comammox were observed in five N gradients (0, 90, 150, 210, and 270 kg ha−1) in a semi-arid area of Northeast China. The results indicated that, compared with low N application rates, higher N application rates significantly improved the soil ammonia monooxygenase (AMO) and the hydroxylamine oxidase (HAO) activities while total nitrogen (TN) was noted to be the main factor driving AMO and HAO activities. Soil potential nitrification rate (PNR) significantly increased with the increase in N application rate, and soil PNR was positively correlated with TN, nitrate nitrogen (NO3−-N), soil organic matter (SOM), and ammonium nitrogen (NH4+-N). The structural equation model (SEM) showed that TN was the main factor driving the community composition of Comammox; SOM had a greatest effect on the community composition of AOA while NH4+-N had a greatest effect on the community composition of AOB. The soil PNR had a direct effect on the yield. Overall, the findings of this study highlighted that N application was more conducive to the reproduction of soil nitrifying microorganisms while Comammox was the dominant contributor to nitrification under N fertilization regimes in the semi-arid area of Northeast China.

Responses of various carbon to nitrogen ratios to microbial communities, kinetics, and nitrogen metabolic pathways in aerobic granular sludge reactor

The role of different ammonia concentrations (mg N/L) (of 100 (carbon to nitrogen ratio (C/N) = 12; Stage I), 200 (C/N = 6; Stage II), 400 (C/N = 3; Stage III) and 200 (C/N = 6; Stage IV)) in nitrogen metabolic pathways, microbial community, and specific microbial activity were investigated in an aerobic granular sludge reactor. Heterotrophic ammonia oxidizing bacteria (HAOB) showed higher ammonia oxidation rates (AORs) than autotrophic ammonia oxidizing bacteria (AAOB) at higher C/N conditions (Stages I and II). Paracoccus was the dominant HAOB. AAOB, with only 0.2–0.3 % in relative abundance, showed 2.7-fold higher AORs than HAOB at elevated ammonia and free ammonia (FA) concentrations with C/N at 3. Nitrosomonas and a genus in Nitrosomondaceae family were the major AAOB. This study proposed that FA inhibition on heterotrophic bacteria might be the mechanism that contributes to the development of the autotrophic ammonia oxidation pathway and enrichment of AAOB.

Effects of media length on biofilms and nitrification in moving bed biofilm reactors

Biofilms grown on free-floating plastic media are increasingly being used to cultivate biofilms in integrated fixed film activated sludge (IFAS) and moving bed bioreactor (MBBR) systems for wastewater treatment with the common goal of increasing nitrogen removal. Fundamental principles of fluid dynamics dictate that the length of internal media channels affects fluid velocities and shear forces across biofilm surfaces, which in turn should affect rates of mass transfer and biofilm growth and activity, but little is known about media length effects on water quality and biofilm characteristics. It was hypothesized that length affects biofilm thickness, microbial populations and their activities, and system performance. Nitrification rates and biofilm characteristics were monitored in parallel continuous flow, bench-scale MBBRs systems with media length as a controlled variable. Longer media produced biofilms with approximately twice the thickness and twice the mass per unit area than did media with one-third their length. Based on calculated head losses, the combined effects of length and constriction of internal channels led to an estimated 77% reduction in fluid velocity through the longer media relative to the shorter media. Longer media demonstrated more rapid development of nitrite oxidizing bacteria (NOB) activity than the shorter media over much of the study, as indicated by measurements of nitrite and nitrate, but AOB activity was similar in the two media. Both biomass and NOB activity were concentrated toward media ends, while ammonia oxidizing bacteria (AOB) activity was uniformly distributed across the media, based on testing of sectioned media. 16s rRNA amplicon sequencing indicated the presence of several putative heterotrophic nitrifying families, particularly Xanthomonadaceae, Comamonadadeae and Microbacteriaceae, as well as the autotrophic Bradyrhizobiacea (which includes the NOB Nitrobacter) were common on both media throughout the study. The short media enriched for Nitrosomonadaceae, which includes the AOB genus Nitrosomonas, while minimal autotrophic AOBs were found in the long media biofilm. These results provide insights to the design of media for improved performance, particularly with respect to nitrite versus nitrate production, which may be useful to improve nitrification and for energy saving processes for nitrogen removal such as deammonification. The research also provides fundamental insights regarding the effects of media geometry on biofilm structure and function, which advances our understanding of environmental factors affecting biofilm development.

Ammonia Nitrogen Removal Performance with Parallel Operation of Conventional and Inverted A2/O Sewage Treatment Processes in Winter

Ammonia nitrogen (NH4+-N) removal capacities of the A2/O and inverted A2/O processes were analyzed with the same inlet and parallel operation during winter. When the operating water temperature was 14℃, the inverted A2/O process exhibited lower NH4+-N removal from the volumetric load[0.13 kg ·(m3 ·d)-1vs. 0.29 kg ·(m3 ·d)-1] and a lower ammonia oxidation rate (AOR)[0.07 kg ·(kg ·d)-1 vs. 0.11 kg ·(kg ·d)-1] than the A2/O process, whereas the two processes exhibited similar performance at 26℃.The quantitative results for the ammonia oxidizing bacteria (AOB) population were almost the same in the two parallel processes (3.2%±0.24% for the inverted A2/O process and 3.4%±0.31% for the A2/O process). Clone library analysis showed that at low temperatures, the inverted A2/O process had a lower capacity for ammonia nitrogen removal than A2/O process. This is because the particular AOB species[spirillum (Nitrosospira)] facilitated the slower AOR type (K-growth strategy) of nitrosation in the inverted A2/O process, whereas in the A2/O process, the faster AOR type (r-growth strategy) of nitrosation was facilitated by bacterium (Nitrosomonas). At 26℃, the dominant species in the two processes were Nitrosomonas. Through comprehensive analysis of the pollutants during the removal process, we found that although temperature is the leading cause of AOB advantage in species succession, the changes in the inverted A2/O process structure, caused by the aerobic unit, resulted in high COD load and high NH4+-N concentration, which were unfavorable for the growth of AOB. This shows that under conventional sewage conditions, the K-growth strategy is advantageous for the AOB species. Therefore, the structure of the inverted A2/O process for heterotrophic bacteria (phosphorus accumulating bacteria and denitrifying bacteria) indirectly affects the population distribution and succession of autotrophic ammonia-oxidizing bacteria, through COD load and other factors, thereby leading to weakened nitrification capacity at low temperatures.

Roles of Organic Matter-Induced Heterotrophic Bacteria in Nitritation Reactors: Ammonium Removal and Bacterial Interactions

The bloom of heterotrophic bacteria resulting from the presence of organic matter in wastewater streams can potentially affect the growth and activity of autotrophic ammonia-oxidizing bacteria (AOB...

DNA stable isotope probing revealed no incorporation of 13CO2 into comammox Nitrospira but ammonia-oxidizing archaea in a subtropical acid soil

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. Purpose: The recently discovered complete ammonia oxidizers (comammox Nitrospira) challenged our concept of the whole nitrification process, i.e., ammonia oxidation catalyzed by autotrophic ammonia-oxidizing archaea (AOA) and bacteria (AOB), followed by the oxidation of nitrite to nitrate. However, the relative contributions of different ammonia oxidizers to soil nitrification are poorly understood. Materials and methods: Soil samples were collected from five land use types (i.e., cropland, grassland, bushland, transition land, and forest land) from a subtropical soil in Yunnan Province, Southwest China. The quantitative distribution of comammox Nitrospira, AOA and AOB in soils was firstly investigated using the QPCR approach and three of which (cropland, transition land and forest land) with pH below 6 were further examined using DNA stable isotope probing approach with 13CO2. Clone library combined with sequencing was applied to detect the phylogenetic information of active microbial groups. Results and discussion: The results showed that the amoA gene abundance of ammonia oxidizers except AOB was significantly lower in croplands than other land uses. Based on the 13CO2 labeling method, AOA, rather than AOB or comammox Nitrospira, was found to incorporate 13CO2 into their genomes during the incubation in cropland and transition land but not in forest land, suggesting the dominant role of AOA in ammonia oxidation. Phylogenetic analysis of the active AOA group revealed that autotrophic AOA community was mainly affiliated with the cluster of Nitrososphaera in transition land, whereas Nitrososphaera, Nitrosopumilus, and Nitrosotalea in cropland. Conclusions: These findings suggested niche differentiation of AOB, AOA, and comammox Nitrospira in the subtropical acid soil with different land uses, and AOA played a more important role in nitrification process of this acid soil.

Nitrogen removal pathway and dynamics of microbial community with the increase of salinity in simultaneous nitrification and denitrification process

In this study, simultaneous nitrification and denitrification (SND) process was successfully established in a hybrid sequencing batch biofilm reactor (HSBBR). High removal efficiency of NH4+-N (98.0±2.4% to 99.8±0.4%) and COD (86.6±4.0% to 91.6±1.8%) was observed in the salinity range of 0.0 to 2.4%. SND via nitrite, replacing SND via nitrate, became the main nitrogen removal pathway at 1.6% and 2.4% salinity. Suspended sludge and biofilm shared similar microbial composition. Dominant genera were substituted by salt-adaptable microbes as salinity increasing. Abundance of autotrophic ammonia-oxidizing bacteria (Nitrosomonas) increased with elevated salinity, while autotrophic nitrite-oxidizing bacteria (Nitrospira) exhibited extreme sensitivity to salinity. The presence of Gemmata demonstrated that heterotrophic nitrification co-existed with autotrophic nitrification in the SND process. Aerobic denitrifiers (Denitratisoma and Thauera) were also identified. Thiothrix, Sedimenticola, Sulfuritalea, Arcobacter (sulfide-based autotrophic denitrifier) and Hydrogenophaga (hydrogen-based autotrophic denitrifier) were detected in both S-sludge and biofilm. The occurrence of ANAMMOX bacteria Pirellula and Planctomyces indicated that ANAMMOX process was another pathway for nitrogen removal. Nitrogen removal in the HSBBR was accomplished via diverse pathways, including traditional autotrophic nitrification/heterotrophic denitrification, heterotrophic nitrification, aerobic and autotrophic denitrification, and ANAMMOX.

The role of ammonium oxidising bacteria (AOB) in ionic liquid 1-dodecylpyridinium chloride removal.

Ionic liquids (IL) have emerged as the next-generation "green" solvent that can replace traditional organic solvent due to properties such as high thermal stability and no vapour pressure. However, their increased usage inevitably allows them to find their way into the environment. The objective of this study was to evaluate the role of autotrophic ammonia-oxidising bacteria (AOB) in the potential removal of 1-dodecylpyridinium chloride ([DPy]+Cl) in both short- and long-term studies. In short-term batch experiments, it was observed that a notable amount of [DPy]+ can be removed by the AOB culture with the removal mechanism being biodegradation and absorption, with the latter playing a greater role. It was also found that [DPy]+ can be released back into the liquid phase when AOB's preferred substrate, NH3, was present. In the long-term study, [DPy]+Cl was successfully biodegraded and a total of nine transformation products were identified. The biodegradation pathway was also proposed. This study demonstrated that [DPy]+Cl can be biological transformed by enriched AOB culture and the accumulation of the by-product did not show long-term negative impact on AOB activities.

Nitrogen cycling during wastewater treatment.

Many wastewater treatment plants in the world do not remove reactive nitrogen from wastewater prior to release into the environment. Excess reactive nitrogen not only has a negative impact on human health, it also contributes to air and water pollution, and can cause complex ecosystems to collapse. In order to avoid the deleterious effects of excess reactive nitrogen in the environment, tertiary wastewater treatment practices that ensure the removal of reactive nitrogen species need to be implemented. Many wastewater treatment facilities rely on chemicals for tertiary treatment, however, biological nitrogen removal practices are much more environmentally friendly and cost effective. Therefore, interest in biological treatment is increasing. Biological approaches take advantage of specific groups of microorganisms involved in nitrogen cycling to remove reactive nitrogen from reactor systems by converting ammonia to nitrogen gas. Organisms known to be involved in this process include autotrophic ammonia-oxidizing bacteria, heterotrophic ammonia-oxidizing bacteria, ammonia-oxidizing archaea, anaerobic ammonia oxidizing bacteria (anammox), nitrite-oxidizing bacteria, complete ammonia oxidizers, and dissimilatory nitrate reducing microorganisms. For example, in nitrifying-denitrifying reactors, ammonia- and nitrite-oxidizing bacteria convert ammonia to nitrate and then denitrifying microorganisms reduce nitrate to nonreactive dinitrogen gas. Other nitrogen removal systems (anammox reactors) take advantage of anammox bacteria to convert ammonia to nitrogen gas using NO as an oxidant. A number of promising new biological treatment technologies are emerging and it is hoped that as the cost of these practices goes down more wastewater treatment plants will start to include a tertiary treatment step.

Effects of salinity on nitrification efficiency and bacterial community structure in a nitrifying osmotic membrane bioreactor

Abstract The objective of this study was to evaluate the effects of salt accumulation on nitrifying bacterial communities in a nitrifying bioreactor combined with forward osmosis. The conversion of nitrite to nitrate was inhibited at a total dissolved solids (TDS) concentration of 17.3 g/L, whereas conversion of ammonia to nitrite was inhibited at a higher concentration (52.8 g-TDS/L). The gene copies of ammonia-oxidizing bacteria (AOB) were more abundant than those of nitrite-oxidizing bacteria (NOB) throughout the entire operating period of 225 days. Among NOB, the number of copies of Nitrobacter spp. were 100–1000 times higher than those of Nitrospira spp. A total of 140 operational taxonomic units were identified using 454 pyrosequencing. The relative abundances of autotrophic AOB and NOB accounted for 34.1–57.8% during 225 days. Dominance of Nitrosomonas eutropha was stable as a salt-tolerant AOB, but the representative NOB, Nitrobacter winogradskyi, showed salt-sensitive variations in their relative abundance. Nonmetric multidimensional scaling and hierarchical clustering analysis clearly illustrated the shift in bacterial community due to external conditions, i.e., ammonia loading rate, alkalinity availability, and salinity. Heterotrophic bacteria contributed to changes in overall bacterial community structure in the nitrifying osmotic membrane bioreactor despite the absence of carbon sources in the influent.

Bacterial community structure in simultaneous nitrification, denitrification and organic matter removal process treating saline mustard tuber wastewater as revealed by 16S rRNA sequencing

A simultaneous nitrification, denitrification and organic matter removal (SNDOR) process in sequencing batch biofilm reactor (SBBR) was established to treat saline mustard tuber wastewater (MTWW) in this study. An average COD removal efficiency of 86.48% and total nitrogen removal efficiency of 86.48% were achieved at 30gNaClL−1 during 100days’ operation. The underlying mechanisms were investigated by PacBio SMRT DNA sequencing (V1–V9) to analyze the microbial community structures and its variation from low salinity at 10gNaClL−1 to high salinity at 30gNaClL−1. Results showed elevated salinity did not affect biological performance but reduced microbial diversity in SBBR, and halophilic bacteria gradually predominated by succession. Despite of high C/N, autotrophic ammonia-oxidizing bacteria (AOB) Nitrosomonas and ammonia-oxidizing archaea (AOA) Candidatus Nitrososphaera both contributed to ammonium oxidation. As salinity increasing, nitrite-oxidizing bacteria (NOB) were significantly inhibited, partial nitrification and denitrification (PND) process gradually contributed to nitrogen removal.

Metabolism and Growth of Autotrophic Ammonia Oxidizing Bacteria with Hydroxylamine as the Sole Energy and Nitrogen Source

Metabolism and Growth of Autotrophic Ammonia Oxidizing Bacteria with Hydroxylamine as the Sole Energy and Nitrogen Source

Microbial Diversity of Ammonia Oxidizing Bacteria through Waste Water Genomics

Autotrophic ammonia-oxidizing bacteria are a critical factor of the microbial community in industrial wastewater treatment systems. We evaluated the diversity and community composition of β-proteobacterial ammonia-oxidizing bacteria in two full-scale treatment reactors - a sand filter and a biological aerated filter - receive an identical wastewater. Polymerase chain reaction of the 16S rRNA gene fragments of ammonia-oxidizing bacteria-selective primers was merged with denaturing gradient gel electrophoresis to allow the comparative analysis of the dominant ammonia-oxidizing bacteria populations. The phylogenetic affinities of the dominant ammonia-oxidizing bacteria were verified by cloning and sequencing of polymerase chain reaction-amplified 16S rRNA gene fragments. Denaturing gradient gel electrophoresis profiles were evaluated using a probability-based similarity index. An exploitation of a probabilistic index of similarity permitted us to consider the differences and similarities observed in ammonia-oxidizing bacteria community structure in different samples were statistically significant or could be accounted for random matching of bands in denaturing gradient gel electrophoresis profiles that would propose random colonization of the reactors at different ammonia-oxidizing bacteria. All Possibly-like sequences recognized, grouped within the Nitrosomonas genus. A greater diversity of ammonia-oxidizing bacteria were detected in trickling filters than the BAF on all samples analyzed were initiate to be dominated by ammonia oxidizing bacteria most closely linked to Nitrosococcus mobilis. Numerical investigation of the denaturing gradient gel electrophoresis profiles indicated that the ammonia-oxidizing bacteria community in depth profiles from the filter beds was selected in a non-random manner.

Predation of nitritation-anammox biofilms used for nitrogen removal from wastewater.

Predation is assumed to be a major cause of bacterial mortality in wastewater treatment plants (WWTP). Grazing on the slowly growing autotrophic ammonia oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AMX) may result in loss of biomass, which could compromise nitrogen removal by the nitritation-anammox process. However, predation, particularly of anaerobic AMX, is unknown. We investigated the presence of protozoa, AOB and AMX and the possible predation in nitritation-anammox biofilms from several WWTPs. By fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM), predator and prey were localized in intact biofilm cryosections. Different broad morphological types of protozoa were found at different biofilm depths. Large variations in abundance of protozoa were seen. One reactor showed a predation event of amoeba-like protozoa, forming grazing fronts reaching deep biofilm regions that were dominated by the anaerobic AMX. Both AOB and AMX were grazed by the amoeba, as revealed by FISH-CLSM. Hence, even AMX, living in the deeper layers of stratified biofilms, are subjected to predation. Interestingly, different colocalization was observed between the amoeba-like protozoa and two different Ca. Brocadia AMX sublineages, indicating different grazing patterns. The findings indicate that predation pressure can be an important factor regulating the abundance of AOB and AMX, with implications for nitrogen removal from wastewater.

Isolation of Ammonia Oxidizing Bacteria (AOB) from Fish Processing Effluents

In the present study, autotrophic ammonia oxidizing bacteria (AOB) were isolated and characterized from a fish processing effluent treatment plant. Sequencing of 16S rRNA gene showed AOB affiliation to Nitrosomonas marina, Nitrosomonas nitrosa and Nitrosospira lineages of Betaproteobacteria. The isolates were further confirmed with using ammonia mono-oxygenase subunit-A gene (amoA) specific fluorescence in situ hybridization assay. The reduction of ammonia by these isolates was comparable with the standard AOB pure culture Nitrosomonas europaea ATCC 19718. Among all the cultures, AOB-21 was found to be having highest potential for ammonia removal. The presence of AOB in fish processing effluent treatment plants suggests the usefulness of these isolates in sustainable removal of ammonia from fish processing waste effluent.

Bacterial community compositions of coking wastewater treatment plants in steel industry revealed by Illumina high-throughput sequencing

In this study, Illumina high-throughput sequencing was used to reveal the community structures of nine coking wastewater treatment plants (CWWTPs) in China for the first time. The sludge systems exhibited a similar community composition at each taxonomic level. Compared to previous studies, some of the core genera in municipal wastewater treatment plants such as Zoogloea, Prosthecobacter and Gp6 were detected as minor species. Thiobacillus (20.83%), Comamonas (6.58%), Thauera (4.02%), Azoarcus (7.78%) and Rhodoplanes (1.42%) were the dominant genera shared by at least six CWWTPs. The percentages of autotrophic ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were unexpectedly low, which were verified by both real-time PCR and fluorescence in situ hybridization analyses. Hierarchical clustering and canonical correspondence analysis indicated that operation mode, flow rate and temperature might be the key factors in community formation. This study provides new insights into our understanding of microbial community compositions and structures of CWWTPs.

Modeling of Nitrous Oxide Production by Autotrophic Ammonia-Oxidizing Bacteria with Multiple Production Pathways

Autotrophic ammonia oxidizing bacteria (AOB) have been recognized as a major contributor to N2O production in wastewater treatment systems. However, so far N2O models have been proposed based on a single N2O production pathway by AOB, and there is still a lack of effective approach for the integration of these models. In this work, an integrated mathematical model that considers multiple production pathways is developed to describe N2O production by AOB. The pathways considered include the nitrifier denitrification pathway (N2O as the final product of AOB denitrification with NO2(-) as the terminal electron acceptor) and the hydroxylamine (NH2OH) pathway (N2O as a byproduct of incomplete oxidation of NH2OH to NO2(-)). In this model, the oxidation and reduction processes are modeled separately, with intracellular electron carriers introduced to link the two types of processes. The model is calibrated and validated using experimental data obtained with two independent nitrifying cultures. The model satisfactorily describes the N2O data from both systems. The model also predicts shifts of the dominating pathway at various dissolved oxygen (DO) and nitrite levels, consistent with previous hypotheses. This unified model is expected to enhance our ability to predict N2O production by AOB in wastewater treatment systems under varying operational conditions.

Effects of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on abundance and activity of ammonia oxidizers in soil

Recent evidence from several environments suggest that besides autotrophic ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) are also able to perform the oxidation of NH4 + to NO2 −, although the relative importance of AOA in nitrification, compared to AOB, and their differential susceptibility to inhibitory compounds remains unclear. Experimental microcosms were set up to evaluate the effect of the addition of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) combined with a cattle effluent as organic fertilizer on the abundance and expression of ammonia oxidizers, denitrifiers, and non-target microbial populations using reverse transcription–real-time PCR, as well as on the diversity of metabolically active soil bacterial and archaeal communities by terminal restriction fragment length polymorphism. While no significant changes in soil mineral N concentrations or amoA gene copies could be detected between treatments, short-term changes in transcriptional activity revealed that DMPP impaired both bacterial and archaeal amoA mRNA, being significant at every time point for AOB and at one time point for AOA. Our findings revealed that, despite the different cellular biochemistry and metabolism existing between bacteria and archaea domains, DMPP exerts its inhibitory effect against both soil bacterial and archaeal ammonia-oxidizing transcriptional activity.

Mutualism between autotrophic ammonia-oxidizing bacteria (AOB) and heterotrophs present in an ammonia-oxidizing colony

Coexistence of an autotrophic ammonia-oxidizing bacterium (Nitrosomonas sp. RA) and heterotrophic bacteria was consistently observed when cultured in an inorganic medium without any external supply of organic carbon. The present study was undertaken to understand the association between autotrophs and the associated heterotrophs for which a system containing active autotrophs and heterotrophs controlled by Hg(2+) addition was developed. The study revealed interdependence of heterotrophs and Nitrosomonas sp. RA for growth under iron-limited condition. Growth of Nitrosomonas sp. RA was supported by siderophores produced by the associated heterotroph, Pusillimonas sp., thereby complementing its high iron requirement while the organics (such as pyruvate) excreted by Nitrosomonas sp. RA during its autotrophic growth supported the survival of heterotrophs in the inorganic medium. The study thus sheds light on the nature of the mutual interactions between heterotrophs and autotrophs that play a role in the ammonia-oxidizing system involved in wastewater treatment.