Active Autotrophic Ammonia-Oxidizing Bacteria in Biofilm Enrichments from Simulated Creek Ecosystems at Two Ammonium Concentrations Respond to Temperature Manipulation

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The Grand River (Ontario, Canada) is impacted by wastewater treatment plants (WWTPs) that release ammonia (NH 3 and NH 4 ؉ ) into the river. In-river microbial communities help transform this ammonia into more oxidized compounds (e.g., NO 3 ؊ or N 2 ), although the spatial distribution and relative abundance of freshwater autotrophic ammonia-oxidizing prokaryotes (AOP) are not well characterized. This study investigated freshwater N cycling within the Grand River, focusing on sediment and water columns, both inside and outside a WWTP effluent plume. The diversity, relative abundance, and nitrification activity of AOP were investigated by denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and reverse transcriptase qPCR (RT-qPCR), targeting both 16S rRNA and functional genes, together with activity assays. The analysis of bacterial 16S rRNA gene fingerprints showed that the WWTP effluent strongly affected autochthonous bacterial patterns in the water column but not those associated with sediment nucleic acids. Molecular and activity data demonstrated that ammonia-oxidizing archaea (AOA) were numerically and metabolically dominant in samples taken from outside the WWTP plume, whereas ammonia-oxidizing bacteria (AOB) dominated numerically within the WWTP effluent plume. Potential nitrification rate measurements supported the dominance of AOB activity in downstream sediment. Anaerobic ammonia-oxidizing (anammox) bacteria were detected primarily in sediment nucleic acids. In-river AOA patterns were completely distinct from effluent AOA patterns. This study demonstrates the importance of combined molecular and activity-based studies for disentangling molecular signatures of wastewater effluent from autochthonous prokaryotic communities.T he Grand River is the largest watershed in Southwestern Ontario, draining into Lake Erie, and is impacted by ammonia (NH 3 and NH 4 ϩ ) sources that directly affect water quality. The oxidation of anthropogenic ammonia to less toxic forms (e.g., NO 3 Ϫ or N 2 ) by ammonia-oxidizing prokaryotes (AOP) helps prevent ammonia from exceeding toxicity thresholds for aquatic organisms and limits for downstream drinking water intake. AOP include aerobic ammonia-oxidizing bacteria (AOB) and archaea (AOA) and anaerobic ammonia-oxidizing (anammox) bacteria.Nitrification is composed of two biological processes: ammonia and nitrite oxidation. Ammonia oxidation is considered the rate-limiting step in nitrification (1) and was believed for many decades to be mediated solely by a group of chemolithoautotrophic AOB (2). In 2005, AOA were also discovered (3). Although studies have confirmed the presence and abundance of AOA in both natural (e.g., references 4-6) and engineered (e.g., references 7-9) environments, relative contributions to ammonia oxidation in these environments are difficult to assess. Although AOA, rather than AOB, were active in soil microcosms and acid soils due to low ammonia availability and their kinetic constant (K m ) for ammonia (10), AOB showed e...

Section: Resultssupporting

confidence: 73%

Wastewater Effluent Impacts Ammonia-Oxidizing Prokaryotes of the Grand River, Canada

Sonthiphand

Cejudo

Schiff

et al. 2013

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“…Stable isotope probing (SIP) with 13 CO 2 has been used to study ammonia oxidizers (14) and, more recently, to distinguish between the relative autotrophic growth of AOB and AOA (15-19). Recently, it was shown for freshwater biofilms that AOB were actively assimilating 13 CO 2 at three different temperatures, whereas labeling of AOA was not observed (20).It is well known that temperature substantially affects the rate of biological reactions. With respect to nitrification, the rate in various environments increases gradually from Ͻ10°C to 30°C (21,22), reflecting the control of temperature on nitrifiers.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Xia

Hernández

et al. 2013

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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, [13 C]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 13 C labeling of ammonia-oxidizing archaea in microcosms incubated at 4 to 25°C was minor. In contrast, significant 13 C 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 ammoniaoxidizing archaea under warmer conditions. I nland waters supply essential ecosystem services to human populations and meanwhile are exposed to anthropogenic disturbances. One outcome of human activity is an increasing load in lakes of nutrients such as nitrogen (N), which together with phosphorus has been shown to be the limiting factor for primary production in aquatic ecosystems (1). With a doubling of the rate of nitrogen input into terrestrial environments (2), nitrification coupled with denitrification is of special significance for sustaining the nutrient balance in freshwater lake ecosystems by returning nitrogen into the atmosphere (3).As the first and rate-limiting step of nitrification, production of nitrite from ammonia is potentially driven by ammonia-oxidizing bacteria (AOB), as well as by ammonia monooxygenase-encoding archaea. These putative ammonia-oxidizing archaea (AOA) have recently been proposed to belong to a new phylum, Thaumarchaeota (4). AOB and AOA occur widely in freshwater habitats (5, 6), raising the question of their relative contributions to nitrification (7). The activity of bacterial ammonia oxidizers has been followed by measuring their pop...

“…4A), contrary to most molecular-method-based studies, which have found Nitrosospira to be the major ammonium-oxidizing genus in agricultural soils (3,4,21,22,26). Fierer et al hypothesized that N additions may result in a shift from a more oligotrophic bacterial community to one that is more copiotrophic, analogous to a shift from K to r selection, terms often used to describe plant species ecology (13).…”

Section: Discussionmentioning

confidence: 64%

“…To stop the nitrification activity, 100 l 1% methiolate (ethylmercurithio-salicylic acid, sodium salt) was added to the subsamples, and the solutions were frozen and kept for chemical analyses. NH 4 ϩ , NO 3 Ϫ , and NO 2 Ϫ concentrations were later measured with a Lachat Autoanalyzer (Lachat Instruments, Milwaukee, WI).…”

Section: Methodsmentioning

confidence: 99%

Impact of Short-Term Acidification on Nitrification and Nitrifying Bacterial Community Dynamics in Soilless Cultivation Media

Cytryn

Levkovitch

Negreanu

et al. 2012

ABSTRACTSoilless medium-based horticulture systems are highly prevalent due to their capacity to optimize growth of high-cash crops. However, these systems are highly dynamic and more sensitive to physiochemical and pH perturbations than traditional soil-based systems, especially during nitrification associated with ammonia-based fertilization. The objective of this study was to assess the impact of nitrification-generated acidification on ammonia oxidation rates and nitrifying bacterial community dynamics in soilless growth media. To achieve this goal, perlite soilless growth medium from a commercial bell pepper greenhouse was incubated with ammonium in bench-scale microcosm experiments. Initial quantitative real-time PCR analysis indicated that betaproteobacterial ammonia oxidizers were significantly more abundant than ammonia-oxidizing archaea, and therefore, research focused on this group. Ammonia oxidation rates were highest between 0 and 9 days, when pH values dropped from 7.4 to 4.9. Pyrosequencing of betaproteobacterial ammonia-oxidizingamoAgene fragments indicated that r-strategist-likeNitrosomonaswas the dominant ammonia-oxidizing bacterial genus during this period, seemingly due to the high ammonium concentration and optimal growth conditions in the soilless media. Reduction of pH to levels below 4.8 resulted in a significant decrease in both ammonia oxidation rates and the diversity of ammonia-oxidizing bacteria, with increased relative abundance of the r-strategist-likeNitrosospira. Nitrite oxidizers (NitrospiraandNitrobacter) were on the whole more abundant and less sensitive to acidification than ammonia oxidizers. This study demonstrates that nitrification and nitrifying bacterial community dynamics in high-N-load intensive soilless growth media may be significantly different from those inin-terraagricultural systems.