Zhongjun Jia

Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil.

Agricultural ecosystems annually receive approximately 25% of the global nitrogen input, much of which is oxidized at least once by ammonia-oxidizing prokaryotes to complete the nitrogen cycle. Recent discoveries have expanded the known ammonia-oxidizing prokaryotes from the domain Bacteria to Archaea. However, in the complex soil environment it remains unclear whether ammonia oxidation is exclusively or predominantly linked to Archaea as implied by their exceptionally high abundance. Here we show that Bacteria rather than Archaea functionally dominate ammonia oxidation in an agricultural soil, despite the fact that archaeal versus bacterial amoA genes are numerically more dominant. In soil microcosms, in which ammonia oxidation was stimulated by ammonium and inhibited by acetylene, activity change was paralleled by abundance change of bacterial but not of archaeal amoA gene copy numbers. Molecular fingerprinting of amoA genes also coupled ammonia oxidation activity with bacterial but not archaeal amoA gene patterns. DNA-stable isotope probing demonstrated CO(2) assimilation by Bacteria rather than Archaea. Our results indicate that Archaea were not important for ammonia oxidation in the agricultural soil tested.

Autotrophic growth of nitrifying community in an agricultural soil

The two-step nitrification process is an integral part of the global nitrogen cycle, and it is accomplished by distinctly different nitrifiers. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, we present the molecular evidence for autotrophic growth of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in agricultural soil upon ammonium fertilization. Time-course incubation of SIP microcosms indicated that the amoA genes of AOB was increasingly labeled by 13CO2 after incubation for 3, 7 and 28 days during active nitrification, whereas labeling of the AOA amoA gene was detected to a much lesser extent only after a 28-day incubation. Phylogenetic analysis of the 13C-labeled amoA and 16S rRNA genes revealed that the Nitrosospira cluster 3-like sequences dominate the active AOB community and that active AOA is affiliated with the moderately thermophilic Nitrososphaera gargensis from a hot spring. The higher relative frequency of Nitrospira-like NOB in the 13C-labeled DNA suggests that it may be more actively involved in nitrite oxidation than Nitrobacter-like NOB. Furthermore, the acetylene inhibition technique showed that 13CO2 assimilation by AOB, AOA and NOB occurs only when ammonia oxidation is not blocked, which provides strong hints for the chemolithoautotrophy of nitrifying community in complex soil environments. These results show that the microbial community of AOB and NOB dominates the nitrification process in the agricultural soil tested.

Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea

The hydrolysis of urea as a source of ammonia has been proposed as a mechanism for the nitrification of ammonia-oxidizing bacteria (AOB) in acidic soil. The growth of Nitrososphaera viennensis on urea suggests that the ureolysis of ammonia-oxidizing archaea (AOA) might occur in natural environments. In this study, 15N isotope tracing indicates that ammonia oxidation occurred upon the addition of urea at a concentration similar to the in situ ammonium content of tea orchard soil (pH 3.75) and forest soil (pH 5.4) and was inhibited by acetylene. Nitrification activity was significantly stimulated by urea fertilization and coupled well with abundance changes in archaeal amoA genes in acidic soils. Pyrosequencing of 16S rRNA genes at whole microbial community level demonstrates the active growth of AOA in urea-amended soils. Molecular fingerprinting further shows that changes in denaturing gradient gel electrophoresis fingerprint patterns of archaeal amoA genes are paralleled by nitrification activity changes. However, bacterial amoA and 16S rRNA genes of AOB were not detected. The results strongly suggest that archaeal ammonia oxidation is supported by hydrolysis of urea and that AOA, from the marine Group 1.1a-associated lineage, dominate nitrification in two acidic soils tested.

Urease gene‐containing Archaea dominate autotrophic ammonia oxidation in two acid soils

The metabolic traits of ammonia-oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea-amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water-amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time-course incubations indicated that archaeal amoA genes were increasingly labelled by (13) CO2 in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the (13) C-DNA, and acetylene inhibition suggests that autotrophic growth of urease-containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a-associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear.

Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils

Rice paddy fields are characterized by regular flooding and nitrogen fertilization, but the functional importance of aerobic ammonia oxidizers and nitrite oxidizers under unique agricultural management is poorly understood. In this study, we report the differential contributions of ammonia-oxidizing archaea (AOA), bacteria (AOB) and nitrite-oxidizing bacteria (NOB) to nitrification in four paddy soils from different geographic regions (Zi-Yang (ZY), Jiang-Du (JD), Lei-Zhou (LZ) and Jia-Xing (JX)) that are representative of the rice ecosystems in China. In urea-amended microcosms, nitrification activity varied greatly with 11.9, 9.46, 3.03 and 1.43 μg NO3−-N g−1 dry weight of soil per day in the ZY, JD, LZ and JX soils, respectively, over the course of a 56-day incubation period. Real-time quantitative PCR of amoA genes and pyrosequencing of 16S rRNA genes revealed significant increases in the AOA population to various extents, suggesting that their relative contributions to ammonia oxidation activity decreased from ZY to JD to LZ. The opposite trend was observed for AOB, and the JX soil stimulated only the AOB populations. DNA-based stable-isotope probing further demonstrated that active AOA numerically outcompeted their bacterial counterparts by 37.0-, 10.5- and 1.91-fold in 13C-DNA from ZY, JD and LZ soils, respectively, whereas AOB, but not AOA, were labeled in the JX soil during active nitrification. NOB were labeled to a much greater extent than AOA and AOB, and the addition of acetylene completely abolished the assimilation of 13CO2 by nitrifying populations. Phylogenetic analysis suggested that archaeal ammonia oxidation was predominantly catalyzed by soil fosmid 29i4-related AOA within the soil group 1.1b lineage. Nitrosospira cluster 3-like AOB performed most bacterial ammonia oxidation in the ZY, LZ and JX soils, whereas the majority of the 13C-AOB in the JD soil was affiliated with the Nitrosomona communis lineage. The 13C-NOB was overwhelmingly dominated by Nitrospira rather than Nitrobacter. A significant correlation was observed between the active AOA/AOB ratio and the soil oxidation capacity, implying a greater advantage of AOA over AOB under microaerophilic conditions. These results suggest the important roles of soil physiochemical properties in determining the activities of ammonia oxidizers and nitrite oxidizers.

Effect of rice plants on CH4 production, transport, oxidation and emission in rice paddy soil

To understand the integrated effects of rice plants (variety Wuyugeng 2) on CH4 emission during the typical rice growth stage, the production, oxidation and emission of methane related to rice plants were investigated simultaneously through laboratory and greenhouse experiments. CH4 emission was significantly higher from the rice planted treatment than from the unplanted treatment. In the rice planted treatment, CH4 emission was higher at tillering stage than at panicle initiation stage. An average of 36.3% and 54.7% of CH4 produced was oxidized in the rhizosphere at rice tillering stage and panicle initiation stage, respectively, measured by using methyl fluoride (MF) technique. In the meantime, CH4 production in the planted treatments incubated under O2-free N2 condition was reduced by 44.9 and 22.3%, respectively, compared to unplanted treatment. On the contrary, the presence of rice plants strongly stimulated CH4 production by approximately 72.3% at rice ripening stage. CH4 emission through rice plants averaged 95% at the tillering stage and 89% at the panicle initiation stage. Based on these results, conclusions are drawn that higher CH4 emission from the planted treatment than from unplanted treatment could be attributed to the function of rice plants for transporting CH4 from belowground to the atmosphere at tillering and panicle initiation stage, and that a higher CH4 emission at tillering stage than at panicle initiation stage is due to the lower rhizospheric CH4 oxidation and more effective transport mediated by rice plants.

Autotrophic Growth of Bacterial and Archaeal Ammonia Oxidizers in Freshwater Sediment Microcosms Incubated at Different Temperatures

ABSTRACT Both bacteria and archaea potentially contribute to ammonia oxidation, but their roles in freshwater sediments are still poorly understood. Seasonal differences in the relative activities of these groups might exist, since cultivated archaeal ammonia oxidizers have higher temperature optima than their bacterial counterparts. In this study, sediment collected from eutrophic freshwater Lake Taihu (China) was incubated at different temperatures (4°C, 15°C, 25°C, and 37°C) for up to 8 weeks. We examined the active bacterial and archaeal ammonia oxidizers in these sediment microcosms by using combined stable isotope probing (SIP) and molecular community analysis. The results showed that accumulation of nitrate in microcosms correlated negatively with temperature, although ammonium depletion was the same, which might have been related to enhanced activity of other nitrogen transformation processes. Incubation at different temperatures significantly changed the microbial community composition, as revealed by 454 pyrosequencing targeting bacterial 16S rRNA genes. After 8 weeks of incubation, [13C]bicarbonate labeling of bacterial amoA genes, which encode the ammonia monooxygenase subunit A, and an observed increase in copy numbers indicated the activity of ammonia-oxidizing bacteria in all microcosms. Nitrosomonas sp. strain Is79A3 and Nitrosomonas communis lineages dominated the heavy fraction of CsCl gradients at low and high temperatures, respectively, indicating a niche differentiation of active bacterial ammonia oxidizers along the temperature gradient. The 13C labeling of ammonia-oxidizing archaea in microcosms incubated at 4 to 25°C was minor. In contrast, significant 13C labeling of Nitrososphaera-like archaea and changes in the abundance and composition of archaeal amoA genes were observed at 37°C, implicating autotrophic growth of ammonia-oxidizing archaea under warmer conditions.

Interactions between Thaumarchaea, Nitrospira and methanotrophs modulate autotrophic nitrification in volcanic grassland soil

Ammonium/ammonia is the sole energy substrate of ammonia oxidizers, and is also an essential nitrogen source for other microorganisms. Ammonia oxidizers therefore must compete with other soil microorganisms such as methane-oxidizing bacteria (MOB) in terrestrial ecosystems when ammonium concentrations are limiting. Here we report on the interactions between nitrifying communities dominated by ammonia-oxidizing archaea (AOA) and Nitrospira-like nitrite-oxidizing bacteria (NOB), and communities of MOB in controlled microcosm experiments with two levels of ammonium and methane availability. We observed strong stimulatory effects of elevated ammonium concentration on the processes of nitrification and methane oxidation as well as on the abundances of autotrophically growing nitrifiers. However, the key players in nitrification and methane oxidation, identified by stable-isotope labeling using 13CO2 and 13CH4, were the same under both ammonium levels, namely type 1.1a AOA, sublineage I and II Nitrospira-like NOB and Methylomicrobium-/Methylosarcina-like MOB, respectively. Ammonia-oxidizing bacteria were nearly absent, and ammonia oxidation could almost exclusively be attributed to AOA. Interestingly, although AOA functional gene abundance increased 10-fold during incubation, there was very limited evidence of autotrophic growth, suggesting a partly mixotrophic lifestyle. Furthermore, autotrophic growth of AOA and NOB was inhibited by active MOB at both ammonium levels. Our results suggest the existence of a previously overlooked competition for nitrogen between nitrifiers and methane oxidizers in soil, thus linking two of the most important biogeochemical cycles in nature.

Methyl-β-cyclodextrin enhanced biodegradation of polycyclic aromatic hydrocarbons and associated microbial activity in contaminated soil

The contamination of soils by polycyclic aromatic hydrocarbons (PAHs) is a widespread environmental problem and the remediation of PAHs from these areas has been a major concern. The effectiveness of many in situ bioremediation systems may be constrained by low contaminant bioavailability due to limited aqueous solubility or a large magnitude of sorption. The objective of this research was to evaluate the effect of methyl-beta-cyclodextrin (MCD) on bioaugmentation by Paracoccus sp. strain HPD-2 of an aged PAH-contaminated soil. When 10% (W/W) MCD amendment was combined with bioaugmentation by the PAH-degrading bacterium Paracoccus sp. strain HPD-2, the percentage degradation of total PAHs was significantly enhanced up to 34.8%. Higher counts of culturable PAH-degrading bacteria and higher soil dehydrogenase and soil polyphenol oxidase activities were observed in 10% (W/W) MCD-assisted bioaugmentation soil. This MCD-assisted bioaugmentation strategy showed significant increases (p < 0.05) in the average well color development (AWCD) obtained by the BIOLOG Eco plate assay, Shannon-Weaver index (H) and Simpson index (lambda) compared with the controls, implying that this strategy at least partially restored the microbiological functioning of the PAH-contaminated soil. The results suggest that MCD-aided bioaugmentation by Paracoccus sp. strain HPD-2 may be a promising practical bioremediation strategy for aged PAH-contaminated soils.

Inhibition of methane oxidation by nitrogenous fertilizers in a paddy soil

Nitrogenous fertilizers are generally thought to have an important role in regulating methane oxidation. In this study, the effect of ammonium on methane oxidation activity was investigated in a paddy soil using urea at concentrations of 0, 50, 100, 200, and 400 μg N per gram dry weight soil (N/g.d.w.s) and ammonium sulfate at concentrations of 0, 50, and 200 μg N/g.d.w.s. The results of this study demonstrate that urea concentrations of 200 μg N/g.d.w.s. and above significantly inhibit methane oxidation activity, whereas no statistically significant difference was observed in methane oxidation activity among soil microcosms with urea concentrations of less than 200 μg N/g.d.w.s after incubation for 27 days. Similar results were obtained in a sense that methane oxidation activity was inhibited only when the ammonium sulfate concentration was 200 μg N/g.d.w.s in soil microcosms in this study. Phylogenetic analysis of pmoA genes showed that nitrogen fertilization resulted in apparent changes in the community composition of methane-oxidizing bacteria (MOB). Type I MOB displayed an increased abundance in soil microcosms amended with nitrogenous fertilizers, whereas type II MOB dominated the native soil. Furthermore, although no statistically significant relationship was observed between pmoA gene and amoA gene abundances, methane oxidation activity was significantly negatively correlated with nitrification activity in the presence of urea or ammonium sulfate. Our results indicate that the methane oxidation activity in paddy soils might be inhibited when the concentration of ammonium fertilizers is high and that the interactions between ammonia and methane oxidizers need to be further investigated.