Progress 01/01/22 to 12/31/22
Outputs
Target Audience: The target audience is the international scientific community, as well as local stake holders, such as farmers, environmental stewards, students, and the general public. Changes/Problems:No major problems or changes in approach have been made. Small adjustments in approach have been made based on reviewer comments of the original project proposal. These primarily include conducting paired metagenome and metatranscriptome analyses, instead of only metatransciptomic analyses. We also opted to conduct more laboratory experiments to better constrain the optimal timing of field sampling campaigns. What opportunities for training and professional development has the project provided?The project has provided training for two undergraduate students, two Ph.D. graduate students, and one postdoctoral scholar. The two undergraduate students learned wet lab methods for soil chemical analyses and microbial activity measurements. They both presented results of their experiments as posters at the Miami Dade College research day. The two graduate students completed comprehensive course work for their Ph.D. program in Year 1, including basic and advanced bioinformatics, molecular biology, and microbial metabolism. They further learned to prepare and conduct comprehensive field sampling campaigns for in situ greenhouse gas flux measurements, and sampling for soil physical and chemical analyses, as well as molecular microbial analyses. They further conducted soil chemical analyses and microcosm-based microbial activity measurements. The postdoctoral scholar participated in a career development workshop, learned new bioinformatics techniques, and presented a poster at the American Society of Microbiology meeting in Washington, DC. How have the results been disseminated to communities of interest?Results of our project have been shared in one published journal article and one conference poster presentation. In addition, two journal articles are currently under revision and will be published during Year 2. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Based on our findings from Year 1, we are now conducting a seasonal cycle analysis of mineralization, nitrification, denitrification, and associated greenhouse gas fluxes in the transition of dry and wet season, that is associated with drastic loss of combined inorganic nitrogen. Our hypothesis is that some nitrate is removed through denitrification, and the remaining part is removed though leaching. Correlation of microbial respiration rates, combined inorganic N budget, in situ greenhouse gas flux measurements, as well as nitrification and denitrification rate measurements should allow us to quantify each component. We will conduct leaching experiments in Year 3 of the project. We will further continue our characterization of mechanistic aspects of ammonia oxidizer metabolism in Year 2, primarily focusing on conditions facilitating nitrifier denitrification activity in AOB, and response of AOA to oxygen limiting conditions in soils. Objective 2: We will continue our metagenomic and metatranscriptomic analyses. Collection of new samples for these analyses are currently under way. We are particularly interested to understand how nitrification, denitrification, and diverse carbon mineralization pathways are activated or repressed throughout the spring-summer transition period. Following detailed analyses of nitrifier communities, we will further continue to analyze our preliminary metagenomic and metatranscriptomic datasets. We will focus on denitrifier communities, as well as microbial carbon mineralization pathways, and how those are activated between dry and wet seasons, as well as cropped and fallow plots. Objective 3: In Year 2, we will conduct nitrification inhibitor experiments in soil microcosms. Given the diversity of ammonia and urea transporters among the different AOA lineages in our soils, it is of interest to better understand how those lineages individually respond to different nitrification inhibitors. The results of these experiments will inform the design of mesocosm experiments to evaluate N leaching, which we then plan to conduct in Year 3.
Impacts
What was accomplished under these goals?
Objective 1: In Year 1 of the project, we aimed to establish a seasonal cycle analysis of linked carbon- and nitrogen cycling activities in the Everglades Agricultural Area soils. Since hiring and onboarding of graduate students did not allow us to commence comprehensive field work until the beginning of 3rd quarter of Year 1, we continued to work up preliminary data and test experimental conditions for activity measurements during a summer sampling. Our preliminary experiments revealed a seasonal signal of microbial respiration and potential nitrification that is likely shaped by the strong seasonal precipitation regime with dry winter and spring periods and wet summer and fall seasons. Results of our summer sampling campaign revealed low nitrification rates in highly saturated soils along with depressed carbon respiration rates and low denitrification rates. However, potential nitrification rates and potential denitrification rates showed that both processes could be stimulated in all but the fallow soil plot, suggesting that microbial mineralization and denitrification were nitrogen-limited in the cropped soils, whereas they were carbon-limited in the fallow plot. A manuscript with a graduate student of the project as lead author is currently under preparation for submission during Year 2 of the project. Both, nitrification and denitrification are responsible for significant N2O emissions from terrestrial systems. In the EAA soils investigated here, preliminary experiments during a seasonal cycle with monthly measurements showed significant N2O emissions only during one sampling campaign in spring. Ammonia oxidation by AOB is thought to contribute significantly to global nitrous oxide (N2O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required for ammonia oxidation. Understanding of the role of NO in the AOB metabolism may improve our ability to evaluate N2O production pathways in agricultural soils. However, tracing NO metabolism at low concentrations remains notoriously difficult. We used a combination of electrochemical sensors and the mild NO scavenger 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. We found that PTIO does not affect NH3 oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea strains unlike inhibitors for ammonia oxidation such as allylthiourea (ATU) and acetylene, although it effectively scavenged NO from the cell suspensions. We next quantified the rate of NO production for N. europaea during O2 replete and O2 limited growth. The results showed that in the absence of nitrifier denitrification, NO production by N. europaea amounted to 3.15% - 6.23% of NO2? production, whereas N. europaea grown under O2 limitation actively denitrified and produced NO equivalent of up to 40% of NO2? production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O2 limitation abolished N2O production. These results indicate different roles of NO during NH3 oxidation and nitrifier denitrification in AOB. The results suggest that, in contrast to nitrifier denitrification, NO may not be a free intermediate or remain tightly bound to iron-centers of enzymes during hydroxylamine oxidation and only NH3 saturation may lead to significant dissociation of NO from hydroxylamine dehydrogenase (HAO). Further experiments with other AOB and AOA strains will be needed, however, our results suggest that NO as a free intermediate may only be relevant under O2 limited conditions in soils. Thus, NO may only be a marker for nitrifier denitrification, but not for general ammonia oxidation metabolism. A manuscript describing the results of these experiments with a graduate student of the project is currently under revision at Applied and Environmental Microbiology. Objective 2: Soil microbial communities are complex with several hundred up to a few thousand active microbial species. Understanding the many interactions and chemical hand-offs within and between microbial functional guilds remains a formidable challenge. To gain a better systemic understanding of these processes and their impact on carbon storage and retention in our soils, we aimed in this project to link process rate measurements (Objective 1) to molecular markers of microbial activity via paired metagenomic and metatranscriptomic analyses of our soils. To understand the dynamic of microbial communities over time in our soils, in Year 1, we first conducted a comprehensive analysis of microbial community structure via 16S rRNA gene amplicon sequencing in our preliminary data seasonal time series. The results showed that overall microbial community structure does not substantially change over a 12-month time period independent of which primer set is used for the analyses. These results corroborate previous studies in other soils, indicating that more detailed metatranscriptomic analyses are needed to evaluate the activity patterns in complex soil microbial communities. A manuscript describing these results in detail with postdoctoral scientist of the project as first author was recently published in Frontiers in Microbiology. Collection of seasonal samples for paired metagenomic and metatranscriptomic analysis is currently under way. In Year 1, we therefore focused on further analyses of paired metagenomic and metatranscriptomic data from our preliminary time series. We first aimed to characterize nitrifier communities. Indeed, soil represents the largest reservoir of Archaea on Earth. The diversity of archaea in soils globally is dominated by members of the class Nitrososphaeria. The evolutionary radiation of this class is thought to reflect adaptations to a wide range of temperature, pH and other environmental conditions. However, the mechanisms that govern competition and coexistence among Nitrososphaeria lineages in soil still remain poorly understood. Analyzing the preliminary data seasonal paired metagenomic and metatranscriptomic data, we found that the predominant soil Nitrososphaeria lineages compose a patchwork of gene inventory and transcription profiles for high- and low affinity ammonia, urea and phosphate utilization pathways. Intriguingly, we found that genes encoding for carbon fixation, respiration and ATP synthesis were conserved and transcriped consistently among predominant phylotypes across 12 major evolutionary lineages commonly found in soil. To our surprise, gene transcription profiles in our soils in situ closely resembled pure culture reference strains under optimal growth conditions. Our results revealed resource-based coexistence patterns among the dominant archaeal lineages in soil, and suggest complementary ecophysiological niches associated to differential nutrient acquisition strategies among globally predominant archaeal nitrifiers in soil. In the context of our soil studies, these results suggest that AOA communities in soils are much more functionally diverse than AOB communities. These findings may have implications on the response of soil nitrification to fertilizer and nitrification inhibitor applications. A manuscript describing the results of these analyses with the postdoctoral scientist as first author is currently under revision at the ISME Journal. Objective 3: No results have been obtained for this Objective in Year 1.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Jun Zhao, Jonathan Rodriguez, Willm Martens-Habbena (2023) Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils. Frontiers in Microbiology 14:359. DIO:10.3389/fmicb.2023.1140487.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Eunkyung Choi, Sana I. Chaudhry, Willm Martens-Habbena (2023) The role of nitric oxide in hydroxylamine oxidation and nitrifier denitrification of ammonia-oxidizing bacteria. Applied and Environmental Microbiology (in revision)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Jun Zhao, Laibin Huang, Seemanti Chakrabarti, Jennifer Cooper, EunKyung Choi, Carolina Ganan, Bryn Tolchinsky, Eric W. Triplett, Samira H. Daroub, Willm Martens-Habbena (2023) Nutrient acquisition strategies drive coexistence patterns among globally predominant archaeal lineages in soil. The ISME Journal (in revision).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Zhao, J., Huang, L. P, Rodriguez, J., Choi, E., Chakrabarti, S., Cooper, J., Daroub, S.H., Martens-Habbena, W. (2022) Microbial gene expression informs differential sensitivity of nitrogen-cycling pathways to environmental change in soil. American Society of Microbiology Microbe Meeting, Washington, D.C. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Rodriguez, J., Zhao, J., Choi, E., Martens-Habbena, W. (2022) Characterization of nutrient-limited nitrifier enrichments in chemostat reactors from two diverse soils. American Society of Microbiology Microbe Meeting, Washington, D.C. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Choi, E., Rodriguez, J., Zhao, J.,Martens-Habbena, W. (2022) Niche differentiation among Nitrosocosmicus sp. affiliated ammonia-oxidizing archaea from soil. American Society of Microbiology Microbe Meeting, Washington, D.C. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Zhao J.P, Huang L. P, Chakrabarti S., Cooper J. p, Choi E. G, Ganan C.U, Tolchinsky B.U, Triplett, E.W., Daroub, S.H., Martens-Habbena W. (2022) Physiological adaptations drive diversity in soil nitrifying communities. International Society of Microbial Ecology Meeting, Lausanne, Switzerland, poster presentation. Poster presentation.
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