Progress 02/01/12 to 01/31/16
Outputs
Target Audience:International and national specialist researchers in the field of nitrification. Soil scientists generally interested in N use in agriculture. Agronomists and field agents with interest in N use efficiency in cereals, grass seed and fodder crops produced in the Pacific North West. Microbiologsts, biochemists and molecular biologists with interest in nitrification. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral associate and one graduate student were paid by this grant. The postdoctoral associate has developed collaborations with international scientists in Austria and UK which have resulted in a publication. She has also initiated correspondence with internationally renowned scientists in Sweden and New Zealand to facilitate model development. The postdoctoral associate has advised a lab technician a graduate student rotation, and three undergraduate students which resulted in the undergraduates and the technician being coauthors on peer reviewed publications and on manuscripts in preparation and review. The postdoctoral associate has obtained experience in writing grant proposals for NIFA, NSF and local agricultural agencies. The postdoctoral associate has given three invited seminars on her work including presentations at the International Conferences on Nitrification in Tokyo, Japan, and Edmonton Can. The graduate student participated in a predoctoral fellowship program in Japan that was sponsored by NSF. As a result we have developed an active collaboration with Japanese scientists at Cho University, Tokyo, where the student participated in experiments to gain expertise using the stable isotope of N and measuring 15N labeled nitrous oxide. The graduate student has published two manuscripts on his research in peer reviewed, and is working on a third and fourth manuscript for submission in 2016. The graduate student has given oral presentations on his work at the International Conference on Nitrification, Edmonton, and at the ASA, Minneapolis. How have the results been disseminated to communities of interest?In addition to the evidence for results dissemination mentioned above, the PI and/or postdoctoral associate have regularly attended the NIFA program directors annual meetings throughout the tenure of the grant and presented either talks or posters on the work. .The PI and postdoctoral research associate have presented talks to Oregon Agricultural Experiment Branch Station personnel at their field days. We have also presented departmental seminars on the work. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have made considerable progress on the objectives of the grant. We have confirmed with pure cultures of ammonia oxidizing archaea that the C8 alkyne, octyne, is a selective inhibitor of AOB and does not affect AOA over the same concentration range. At much higher concentrations we have identified that octyne can inhibit AOA activity but the data suggest that it does so by a completely different mechanism than has been seen before for oxygenase enzymes that use alkynes as substrates.We believe this isproviding us informationthat the mechanism of ammonia oxidation activity by AOA is quite different than of bacteria and should be researched further because of its implications for alternate selective inhibitors of nitrification. This work has been published and involved a collaboration with scientists from the University of Vienna, Austria. The work was sited as of high impact and interest by ASM. A second study associated with objectives 1 and 4 examined if seasonal conditions influenced the relative contributions of AOA and AOB to soil nitrification and also to determine if the properties of nitrification were affected.Inthis study we found that nitrifying activity of AOA wasgreater insummer than wintersoils, and that the summer soil activity was stimulated by the addition of NH4+. We also found that AOBin cropped soils required a much higher level of NH4+ to stimulate nitrifying activity than in non cropped soils. This work has been published andwas also chosen as a highlight paperfor CSA News and has received considerable interest.A study associated with objective 3 was to examine how soil temperature influenced the relative contributions of AOA and AOB to soil nitrification. This study has shown that soil temperature profoundly affects the relative contributions of AOA and AOB to soil nitrification.We have been modeling the data to illustrate that AOB are more active than AOA at nitrifying in cold soils (4C),and that AOA are more active than AOB in warm soils. In addition we have discovered that AOA and AOB activities respond differentially to temperature changes. Modeling activities are now being more successfulas a result of recognizing these differences. A manuscript is in review on this topic. The final topic being studied in the grant is an examination ofthe relative roles of AOB and AOA driven nitrification to nitrous oxide production. We have shown that nitrification can become uncoupled, ie nitrite accumulates under some soil conditions which directlyresults innitrifier dependent nitrous oxide production.We are in the processof tryting to understand what is accounting for the accumulation of nitrite, and modeling the data toidentify therelationship between nitrite levels and nitrous oxide production. AOB are primarily responsible for the nitrite accumulation and N2O production, but AOA can also produce nitrous oxide.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Lu, X., P.J. Bottomley and Myrold, D.D.. 2015. Contributions of ammonia oxidizing archaea and bacteria to soil nitrification in Oregon forest soils. Soil Biol. Biochem. 85: 54-62.
Mellbye, B., P.J. Bottomley and L. Sayavedra-Soto. 2015. Nitrite oxidizing bacterium Nitrobacter winogradskyi produces N acyl homoserine lactone autoinducers. Appl. Environ. Microbiol. 81: 5917-5926.
Taylor, A.E., Taylor, Kc, Tennigkeit, B., Myrold, D.D., Schleper, C., and Wagner, M., and P.J. Bottomley. (2015). Inhibitory effects of C2-C10 1-alkynes on NH3 oxidation in two Nitrososphaera species. Appl. Environ. Microbiol. 81: 1942-1948.
Giguere, A., A. Taylor, D. Myrold, and P.J. Bottomley. 2015. Nitrification responses of soil ammonia oxidizing Archaea and Bacteria to ammonium concentrations Soil Sci. Soc. Am. J.79: 1366-1374.
M. C. Rice, J. M. Norton, F.Valois, A. Bollmann, P. J.Bottomley et al. 2015. Complete genome of Nitrosospira briensis C-128, an ammonia-oxidizing bacterium from agricultural soil. Standards in Genomic Sciences
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Progress 02/01/14 to 01/31/15
Outputs
Target Audience: International and national specialist researchers in the field of nitrification Soil scientists generally interested in nitrogen use in agriculture. Agronomists and field agents with focus on N use efficiency in winte wheat and grass seed production Microbiologists and molecular biologists interested in nitrification Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One postdoctoral associate and one graduate student were paid by this grant. The postdoctoral associate has developed collaborations with international scientists in Austria and UK which have resulted in a publication. She has also initiated a collaboration with a nationally renowned scientist at Utah State University to facilitate model development. The postdoctoral associate advised a lab technician and two undergraduate students which has resulted in the undergraduates and the techician being coauthors on our publications and ona manuscript in preparation. The postdoctoral associate has obtained experience in writing grant proposals for NIFA, NSF and local agricultural agencies. The psotdoctoral associate has given two invited seminars on her work. The graduate student participated in a predoctoral fellowship program in Japan that was sponsored by NSF. As a result we have developed an active collaboration with Japanese scientists at Cho University where the student participated in experiments to gain expertise using the stable isotope of N and measuring labled nitrous oxide. The graduate student has submitted a manuscript on his research to SSSAJ, and is working on a second manuscript for submision in 2015. How have the results been disseminated to communities of interest? The PI attended theNIFA program directors annual meeting in Washington DC in 2014 and presented a talk on the work to date. The postdoctoral research associate presented talks to Oregon Branch Station personnel at their field days. She also presented a departmental seminar on her work. What do you plan to do during the next reporting period to accomplish the goals? Goals for 2015. Determine the impact of soil temperature and NH4+ availability on the contributions of archaea and bacteria to soil nitrification. Identify the specific subpopulations of NH3 oxidizers contributing at different temperatures and their kinetic parameters. Initiate workon a constraints based model of nitrification. Complete work ondetermining the relative rols of bacteria and archaea to nitrifer produced nitrous oxide in cropping systems of Oregon.
Impacts
What was accomplished under these goals?
We have made considerable progress on the objectives of our grant. We have confirmed with pure cultures of ammonai oxidizing archaea that the alkyne, octyne, is a selective inhibtor of AOB and does not affect AOA over the same concentration range. At much higher concentrations we have identified that octyne can inhibit AOA activity but the data suggest that it does so by a completely different mechanism than has been seen before for oxygenase enzymes that use alkynes as substrates. We believe this is telling us information about the emchanism of ammonia oxidation activity by AOA is quite different than of bacteria and should be researched further because of its implications for alternateselective inhibitors of nitrification. Wehavecontinued with our studies to examine the influence of temperature on relative contributions of Archaea and Bacteria to soil nitrification.Studies have been conducted on different soils fromthroughout Oregon.An interesting feature fo this work is to better refine the parameter of temperature in models of nitrification, and also to determine the identityof theammonia oxidizers in the different temperature niches. This is also of particular interest in the context of nitrous oxide production, and in terms of the levels of ammonia -N needed to support nitrification for different groups planted at different seasons of the year ie fall versus spirng planted. We have completed a project examining the response of bacterial and archaeal nitrification to different ammonia applications. Our data have shown that the amounts of ammonia needed to saturate bacterial oxidation is much greater than required to saturate archaeal nitrification, and that there are differences to how archaeal nitrification responds in summer versus winter soils.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Perez, J., A. Buchanan, R. Ferrell, B. Mellbye, J. Chang, F. Chaplen, D. J. Arp, P. J. Bottomley, and L. Sayavedra-Soto. 2014. Interactions of Nitrosomonas europaea and Nitrobacter winogradskyi grown in coculture . Archives of Microbiology. DOI 10.1007/s00203-014-1056-1
Xinda Lu, Peter J. Bottomley, and David D. Myrold (2015). In Press. Contributions of Ammonia-oxidizing Archaea and Bacteria to Nitrification in Oregon Forest Soils. Soil Biology&Biochemistry
Taylor, A.E., et al. (2015) Inhibitory effects of n-alkynes on ammonia oxidation by two Nitrososphaera species. Apll.Environ. Microbiol. 81: doi 10.1128/AEM.03688-14
Sayavedra-Soto, L., R. Ferrell, M. Dobie, R. Ferrell, Mellbye, B., F. Chaplen, A. Buchanan, J. Chang, Arp, D., and P.J. Bottomley. 2014. In press. Nitrobacter winogradskyi transcriptomic response to low and high ammonium concentrations. FEMS Microbiology
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Progress 02/01/13 to 01/31/14
Outputs
Target Audience: International and national specialist researchersin the field of nitrification. Soil scientists generally interested in nitrogen use in agriculture. Agronomists and field agents with focus on N use in winter wheat production. Microbiologists and molecular biologists interested in nitrification. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One postdoctoral associate and one graduate student are paid by this grant. The postdoctoral associate attended the 3rd internatioanl conference on nitrification held at Chuo University in Tokyo,Japan. Shewas invited to presentthe plenary paperin a specialist session. The postdoctoral associate has been given the opportunity to develop international collaborations with three research groups inCanada, UK and Austria, respectively. The postdoctoral associate attended a genomics annotation jamboree at OSU that was organized by ICON networkand was attended by an international group of scientistswho study nitrification. The postdoctoral associate has been given experience in writing grant proposals. The postdoctoral associate has been given the opportunity to advise a lab technician and graduate and undergraduate students. The postdoctoral associate published a paper that was highlighted in Applied and Environmental Microbiology and recommended to the faculty of 1000. The graduate student attended the International conference on nitrification held at Chuo University, Tokyo, Japan. The graduate student attended a graduate student workshop andgave an oral presentation on his research to an international group of his peers and senior scientists at the international conference. The graduate student wrote and submitted an NSF proposal to conduct research in the laboratory of a Japanese investigator during the summer of 2014. How have the results been disseminated to communities of interest? The PI attended the program director's workshop held by NIFA in Annapolis, MD,2013, and presented a poster on the work to date. The PI attended an International conference on Nitrogen Technology at the Technicon University, Haifa, Israel, April, 2013. The co-PI presented two posters on the research at the Annual Meeting of the Soil Science Society of America held in Tampa, FL,Oct. 2013. What do you plan to do during the next reporting period to accomplish the goals? Goals for 2014. Proceed with isotope dilution studies to determine the relationship between gross rates of N mineralization and N immobilization and gross rates of nitrification when conducted by AOA and AOB at different seasons and different soil temperatures. Determine how the relative contributions of AOB and AOA populations to soil nitrification respond to combinations of soil stresses of temperature and water.
Impacts
What was accomplished under these goals?
Outputs. Ammonia oxidizing bacteria (AOB) and ammonia oxidizing thaumarchaea (AOA) co-occupy most agricultural soils. We are conducting research to determine what soil and environmental factors affect their relative contributions to nitrification in agricultural soils. Subsequently, our intent is to determine if there is any impact of these relative contributions on the cycling of N and fertilizer N use efficiency in agricultural soils. We have been conducting a combination of complimentary studies on whole soils and on some recently acquired pure cultures of AOA. We have been making considerable progress on the objectives of this grant. Pure culture studies. With NIFA funding, we have developed a novel method that allows us to distinguish between the contributions of AOB and AOA to in situ soil nitrification in agricultural soils (Taylor et al. 2013). As a result of this work, we have initiated collaborations with researchers of AOA in Canada, UK and Austria. They have provided us with strains of pure cultures of soil –borne AOA to determine if octyne inhibits NH3 oxidation by pure cultures of soil AOA as predicted from our whole soil studies. We have confirmed that the soil AOA isolates are more resistant to octyne than AOB isolates. We have also found that high concentrations of octyne are partially inhibitory to the soil AOA isolates, and that partially inhibited activity remains constant for at least two days. This observation is somewhat unusual because it implies that octyne is inhibiting but not inactivating the AMO of the soil AOA as it does the AMO of AOB,. Studies continue to gain further insight into the mechanism of inhibition by octyne. This result is exciting because it infers that the AMO of soil AOA is quite different from the AMO of AOB, and this should stimulate renewed interest to search for nitrification inhibitors of AOA that are different from those that inhibit AOB. Whole soil assays. Studies are being performed on cropped and non cropped soils using the octyne assay to look at the relative contributions of AOA and AOB, and to determine if there is a relationship with soil extractable or soil solution NH4+ levels. We have created NH3 response profiles for paired cropped and noncropped soils from 4 regions in Oregon. Generally speaking, AOA activity dominates nitrification in noncropped soils without NH4+ additions, while AOB activities dominates in cropped soils amended with NH3 additions. In cropped soils Vmax values of >0.71-1.04 μmol NO3- + NO2- g-1 soil day-1 were achieved with additions of 10-20 µmol NH4+/g soil. Concentrations of NH4+ that support one half the maximum nitrification rates (Ksvalues) ranged between 4.1 ->17.3 μmol KCl extractable NH4+ g-1 soil. AOA activities were measured in cropped soils but were at least 10-fold lower than AOB activity (0.04-0.1 μmol NO3- + NO2- g-1 soil day-1 ), and could not be stimulated by additions of NH4+. In contrast, AOA activities were measured in all noncropped soils and were inconsistently stimulated by low NH3 additions. Maximum AOA activities of noncropped soils were about 3-fold higher than the AOA rates in cropped soils ranging from 0.14-0.26 μmol NO2- + NO3- g-1 soil day-1. In noncropped soils, AOB activities were stimulated by NH3 additions to Vmax values of 0.02-0.91 μmol NO2- + NO3- g-1 soil day-1 and Km values of <0.6-3.5 μmol KCl extractable NH4+ g-1 soil. The latter Ks values are lower than the values measured for AOB in the cropped soils. Studies associated with objectives 1, 2 and 3 of this grant are ongoing to learn why NH3 additions inconsistently stimulate AOA activity under some noncropped soil conditions, and what significance can be attached to the different kinetic properties of AOB in cropped versus noncropped soils. Another whole soil study has been conducted to examine the influence of soil temperature on relative contributions of AOA and AOB. Soil incubations were conducted at different temperatures ranging from 4C to 45C. At temperatures of <10C virtually all of the nitrifying activity was octyne sensitive and attributed to AOB. As the temperature was increased above 15C, AOA activity increased disproportionately more than AOB activity and contributed >60% of activity at temperatures of 30C. Above 30C AOB declined rapidly and all activity ≥35C was due to AOA. These results have been confirmed in two different soils under both cropped and noncropped conditions. Studies are on-going on the potential impact of AOB nitrification in cold soils in response to early spring applications of fertilizer N, and the role of AOA in nitrification in response to the rewet of warm dry soils in the fall. Outcomes. One manuscript has been published (Taylor et al. 2013) in the November 2013 issue of Applied and Environmental Microbiology and was spotlighted as an article of significant interest. It was also recommended as being of special significance in its field by Faculty of 1000. Another manuscript is in preparation where we have refined the assay for use in whole soils and is being prepared for submission to Soil Science Society of America Journal. We anticipate that a manuscript describing the Comparative properties of the pure cultures of AOA will be prepared in early 2014, and will be the first work to report comparative properties of soil AOA cultures. A manuscript describing the results of the different temperature ranges of AOB and AOA activities in the same soils will be ready for submission in 2014.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Vajrala, N., W. Martens-Habbena, L. A. Sayavedra-Soto, A.Schauer, P.J. Bottomley, D.A. Stahl, D.J. Arp. 2013. Hydroxylamine as an intermediate in ammonia oxidation by globally abundant archaea. Proc.Natl.Acad.Sci. 110:1006-1011.
Taylor, A.E., Vajrala, N., Giguere, A., D.J. Arp, L. Sayavedra-Soto, A. Gittelman, D.D. Myrold, and P.J. Bottomley. 2013. Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia oxidizing thaumarchaea and bacteria. Applied and Environmental Microbiology 79: 6544-6551.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Bottomley, P.J. and D.D. Myrold. 2013. Nitrogen inputs to soil. In: Soil Biology and Biochemistry 4rd edition (E.A. Paul, ed). Academic Press.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Invited Speaker, Technicon University, Haifa, Israel. Apr, 2013. Dahlberg Symposium on Newly emerging developments in Nitrogen and Phosphorus Research . Title: Discriminating between archaeal and bacterial contributions to Soil Nitrification.
Invited Speaker. 3rd International conference on nitrification, Tokyo University, Tokyo. Sep 2013. Title: Discriminating between archaela and bacterial contributions to soil nitrification.
Annual presentations at PI workshops for NIFA grant that were held in Annapolis, MD.
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Progress 02/01/12 to 01/31/13
Outputs
OUTPUTS: We are conducting research to assess the soil and environmental factors that affect the contributions of bacteria and thaumarchaea to nitrification rates in agricultural soils. Subsequently, our intent is to determine if there is impact of these contributions on the efficiency of nitrogen fertilizer useage. NH3-oxidizing bacteria (AOB) and NH3 monooxygenase encoding thaumarchaea (AOA) co-occupy most soils. To date, no short term, growth-independent method exists to determine their relative contributions to NH3-oxidizing activity in situ. Based upon literature which shows that microbial monooxygenases differ in their ability to be inactivated by aliphatic n-alkynes of different chain lengths, we found that the rate of NH3-oxidation by the thaumarchaeon, Nitrosopumilus maritimus, was completely unaffected during a 24h exposure to ≤20micromolar n-alkynes C8-C9. In contrast, NH3-oxidation by two species of AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1- 2micromolar of n-alkynes C2-C9. Evidence was obtained that NH3 oxidation carried out by soil borne AOA was also insensitive to octyne (C8). In NH4+ amended, whole soil incubations (21 or 28d) , both acetylene and octyne prevented NH4+ stimulated increases in AOB population densities. In contrast, octyne did not prevent increases in AOA population densities that were prevented by acetylene: furthermore, an octyne-resistant, NH4+-stimulated net nitrification rate of ~2 micrograms N/g/d persisted throughout the incubation. Other evidence that octyne resistant nitrification was due to AOA included: (1) The rates of octyne resistant nitrification in soil slurries of diverse cropped and noncropped Oregon soils correlated positively (r2= 0.938) with the rates of ATU (100 micromolar) resistant activity in the same soil slurries. (2) The fraction of octyne resistant activity in soil slurries correlated with the fraction of nitrification that recovered from acetylene inactivation of slurries within 24-48h in the presence of bacterial protein synthesis inhibitors (r2= 0.905), and, with the octyne resistant fraction of NH4+-saturated net nitrification measured in whole soils (r2 =0.884). The octyne assay should be a versatile, inexpensive method useful for further investigation of the environmental drivers that affect the relative contributions of bacteria and thaumarchaea to soil nitrification. Further work is being focused upon using the octyne method to follow the changes in contributions of AOB and AOA to soil nitrification in response to different levels of NH3 application in diverse Oregon soils under different land uses. Other studies are being initiated that are aimed at elucidating the environmental factors that influence the relative contributions of AOA and AOB in cropped soils. PARTICIPANTS: Dr. Anne Taylor, and Dr. Neeraja Vajrala did the primary work on this project. One graduate student of soil science (Andrew Giguere) has been actively involved for about 1y. Two undergraduates, T. Wanzek, M. Donie) have been involved. Both Dr Taylor and Dr. Vajrala have attended national meetings (ESA and ASM) or PI meetings/workshops (NIFA, 2013). Drs. Bottomley and Myrold have presented aspects of this work at international conferences in Israel and in Denmark. The work has been presented nationally at the Ecological Society of America and at SSSA. Collaborators have included scientists at the Univ. of Aberdeen, Scotland, and Technion University, Haifa, israel. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
One manuscript has been published on this topic (Taylor et al. 2012). Another manuscript describing the above results has been recently submitted for publication in Proceedings of the National Academy of Sciences, USA. We predict that our newly developed octyne assay will be readily adopted by other researchers interested in the factors controlling the rate of nitrification in US agricultural soils. We have used our new assay to show that AOB dominate nitrification in cropped soils throughout Oregon at least when high levels of NH4+ fertilizer are applied. We have shown that most cropped soils possess a high capacity to oxidize NH3 that is only saturated at high N application rates of 140-280 micrograms N/g soil. Some soils seem to be saturated at lower rate of N application which could have practical implications. In contrast, in non cropped soils sampled throughout Oregon, we obtained evidence that AOA dominate nitrification activity, that their activity rate can be stimulated by adding small amounts of NH4+, and that the rate is saturated by a much lower level of applied NH4+ (~14-28 micrograms N/g soil ) than required in the AOB dominated cropped soils. The maximum rates of nitrification supported by AOA are quite consistent lying between 2-5 micrograms N/g/d. In some non-cropped soils we have obtained evidence for contributions of both AOB and AOA to nitrification. We don't yet know what is the difference between soils that have a mixed versus AOA dominated nitrification. The studies extending the octyne assay to whole soils, and to examine the response of nitrification by Archaea and Bacteria to different N rates is being prepared for publication in Soil Biology and Biochemistry.
Publications
- Taylor, A.E.,Zeglin,L.H.,Wanzek,T.A.,Myrold D.D.,and Bottomley,P.J. (2012). Dynamics of ammonia oxidizing archaea and bacteria populations and contributions to soil nitrification potentials. 6:2024-2032.
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