Source: UTAH STATE UNIVERSITY submitted to
FUNCTIONAL GENOMICS AND ECOLOGY OF NITRIFYING BACTERIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
0200470
Grant No.
(N/A)
Project No.
UTA00371
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2010
Project End Date
Jun 30, 2015
Grant Year
(N/A)
Project Director
Norton, J. M.
Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322
Performing Department
Plants, Soils & Climate
Non Technical Summary
Human activities have dramatically altered the global nitrogen cycle by increasing the amount of reactive nitrogen in the environment; human associated inputs of industrially produced N fertilizers and N fixation by crops now exceed the natural N inputs to terrestrial systems. Nitrifying microorganisms play critical roles in the movement of this reactive nitrogen through ecosystems and in the availability of nitrogen for plant growth. In many agricultural soils when available nitrogen supply exceeds plant demand, nitrification increases leading to accumulation of nitrate that is reactive and mobile in the environment. The nitrogen use efficiency of our N fertilizers in agricultural systems remains quite low, typically only around 30% in cereal crops. Nitrification may lead to losses of nitrogen by leaching and denitrification. Nitrate leaching from agricultural systems is a significant contribution to the contamination of surface and groundwater. Nitrification therefore needs to be managed to protect the quality of surface waters and soils. Information is needed on the physiology and ecology of the bacteria and archaea responsible for cycling nitrate in ecosystems. We will improve understanding of the genomics of nitrifiers, characterize the processes in agricultural and wildland systems and examine links between nitrification and plants in soils. Improved understanding of nitrifying bacteria and archaea in soils and in wastewater systems may suggest management options for particular environments. Delineation of the limiting factors for nitrification in water delivery and wastewater treatment systems will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110107030%
1024010107020%
1024010108030%
1330330110020%
Goals / Objectives
Objective 1. Functional genomics of nitrifying bacteria. Supply genomic DNA of selected nitrifying bacteria for draft-level and complete genome sequencing. Work with the Joint Genome Institute and the DOE to facilitate genome sequencing and analysis. Annotate and check functional designations for selected genes especially those encoding enzymatic functions. Perform comparative genome analysis on nitrifying bacteria sequenced. Write collaborative papers. Objective 2. Ecology of nitrification in agricultural systems in the Intermountain West. Collaborate in ongoing agricultural systems experiments comparing conventional and organic fertility management for effects on nitrification. Objective 3. Ecology of nitrification in wildland systems in the Intermountain West. Collaborate in ongoing experiments in wildland systems examining plant species diversity and function to assess nitrifier functional diversity and ecology of nitrification. This area of research will require establishment of collaborative relationships, site assessment and selection and the collection of baseline data on nitrification in a variety of ecosystems in Colorado and Utah. Objective 4. Characterization of N cycling in wastewater treatment systems. Explore wastewater N cycle in selected wastewater treatment systems in Utah. Assess the key microbial communities, specifically ammonia oxidizing bacteria and archaea by the application of molecular tools waste treatment systems. Determine rate parameters for nitrification in lagoon, artificial wetland, and storage pond environments. Identify key vulnerability points of the N cycling processes needed to maintain effluent water quality.
Project Methods
Objective 1. The Norton laboratory will maintain and extend a culture collection of ammonia oxidizing bacteria. Collaborative arrangements through the Nitrification Network (http://nitrificationnetwork.org/) are ongoing and J. Norton will continue as a member of the steering committee. Several genome sequencing projects are in process through the Community Sequencing Program of the Joint Genome Institute. For each bacterium sequenced, a group leader will be identified who will be responsible for organizing the annotation efforts for that bacterium. Much of the work can be done electronically, but we also recognize the desirability of a bringing annotation teams together for an "annotation jamborees". Teams will be established to work on comparative genomic projects. Objective 2. Collaborative arrangements with Dr. Jennifer Reeve will be established to examine the ecology and diversity of nitrifying organisms in agricultural systems research plots, most of these will use UAES research lands. Several ongoing projects will benefit from additional N cycle characterization under organic agricultural management. The community composition of ammonia oxidizing bacteria and archaea will be examined using molecular methods based on the genes encoding the ammonia monooxygenase enzyme. Objective 3. Collaborative arrangements with Prof. John Stark (USU Dept. of Biology) are ongoing to examine the role of belowground feedbacks on the ability of cheatgrass to maintain stable communities (Stability of Weedy Plant Communities in Western Rangelands P.I. J.M. Stark and CoPI J.M.Norton). The study site is located in the Piceance Basin of Northwestern Colorado at 2020 m elevation on deep, well drained, loam textured soils, with a surface soil pH of 7.5. Treatments related to plant community sucession were established in 1984 and therefore the site is extremely valuable for examination of long-term effects of cheatgrass. Soil sampling for N pools and turnover is already incorporated into the experimental measurements; and molecular methods are being applied to examine nitrifier activity and community composition. Methods ammonia oxidizer community composition described under objective 2 will be applied to soils sampled from these sites. Additional possibilities for collaboration include those with: 1) Dr. Tom Monaco and Dr. Doug Johnson (USDA FRRL) to examine plant species composition and N cycle dynamics in a factorial plot experiment following a fire near Malta, Idaho. 2) Dr. Stephen Hart on the the community diversity of nitrifiers in common gardens with cottonwood trees of known genotype planted in 1991 at the Ogden Nature Center, UT. Objective 4. The wastewater municipal treatment facility of the City of Logan, UT will be examined for spatial and temporal interactions of nitrification and denitrification. Additional possibilities exist to examine the smaller lagoon system used by the City of Wellsville, UT, to treat its municipal wastewater (collaborator Prof. Ryan Dupont). One aspect of this system of particular interest is the role of duckweed in removing both nitrogen and phosphorus from the wastewater.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Objective 1. Functional genomics of nitrifying bacteria. Completed finished high quality sequences for Nitrosomonas strains AL212 and IS 79. The genome announcement for Nitrosomonas sp. IS79 has been submitted. Both sequences are available in GenBank; see http://www.ncbi.nlm.nih.gov/genome/1850 and http://www.ncbi.nlm.nih.gov/genome/3155 . Genomic DNA preparation for Nitrosospira briensis and for Nitrosomonas cryotolerans was completed and DNA shipped to the joint Genome Institute for sequencing. Objective 3. Nitrification ecology. Genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea have been amplified from a rangeland site in Colorado. These have been sequenced and analyzed with results released to GenBank. Real-time PCR targeting genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea has been developed and has been completed for both rangeland and agricultural soils. Shifts in the balance between ammonia oxidizing bacteria and ammonia oxidizing archea suggest that these organisms have distinct niches with the ammonia oxidizing archaea favored by undisturbed and low nitrogen conditions in general. PARTICIPANTS: "Marlen Rice (undergraduate student), Brittany Johnson Co-authors: Annette Bollmann Yuichi Suwa, Martin G. Klotz, Lisa Y. Stein, Hendrikus J. Laanbroek and Daniel J. Arp DOE Joint Genome Institute collaborators: Lynne Goodwin, Olga Chertkov, Brittany Held, David Bruce, J. Chris Detter, Janine C. Detter, Roxanne Tapia, and Cliff S. Han" TARGET AUDIENCES: Scientists, students and waste water managers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Improved understanding of nitrifying bacteria and archaea in soils and in wastewater systems may suggest management options for particular environments. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the limiting factors for nitrification in water delivery and wastewater treatment systems will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.

Publications

  • Bollmann, A., Sedlacek, C. J., Norton, J. M., & a. 3. o. (2012) Complete genome sequence of Nitrosomonas sp. Is79 - an ammonia oxidizing bacterium adapted to low ammonium concentrations.: Standards in Genomic Sciences. (Submitted).


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Objective 1. Functional genomics of nitrifying bacteria. Complete and finished high quality sequences were completed for Nitrosomonas strains AL212 and IS 79. The genome announcement for Nitrosomonas sp. AL212 has been published and the genome announcement for Nitrosomonas sp. IS79 is in preparation. Both sequences are available in GenBank; see http://www.ncbi.nlm.nih.gov/genome/1850 and http://www.ncbi.nlm.nih.gov/genome/3155) . Genomic DNA preparation for Nitrosospira briensis and for Nitrosomonas cryotolerans is progressing. Objective 3. Nitrification ecology. Genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea have been amplified from a rangeland site in Colorado. These have been sequenced and analyzed with results released to GenBank. Real-time PCR targeting genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea has been developed and has been completed for both rangeland and agricultural soils. Shifts in the balance between ammonia oxidizing bacteria and ammonia oxidizing archea suggest that these organisms have distinct niches with the ammonia oxidizing archaea favored by undisturbed and low nitrogen conditions in general. Book chapter reviewing the distribution and diversity of ammonia oxidizing bacteria published. Book chapter on nitrogen mineralization and immobilization turnover published. PARTICIPANTS: Eric Cram (undergraduate student) Co-authors: Yuichi Suwa, Annette Bollmann, Martin G. Klotz, Lisa Y. Stein, Hendrikus J. Laanbroek and Daniel J. Arp DOE Joint Genome Institute collaborators: Lynne Goodwin, Olga Chertkov, Brittany Held, David Bruce, J. Chris Detter, Janine C. Detter, Roxanne Tapia, and Cliff S. Han TARGET AUDIENCES: Scientists, educators and government agencies interested in nitrification and management of nitrogen cycling. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Improved understanding of nitrifying bacteria and archaea in soils and in wastewater systems may suggest management options for particular environments. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the limiting factors for nitrification in water delivery and wastewater treatment systems will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.

Publications

  • UTAO+0371 Norton, J. M., 2011. Nitrification, In B.B. Ward, D.J. Arp, M.G. Klotz (Ed.), Diversity and environmental distribution of ammonia-oxidizing bacteria. ASM Press, Washington D.C., p. 39-55. (Published).
  • UTAO+0371 Norton, J. M., & Schimel, J. P., 2011. Handbook of Soil Science, In Pan Ming Huang; Yuncong Li; Malcolm E. Sumner (Ed.), Chapter 27.2 Nitrogen Mineralization Immobilization Turnover. CRC Press, Boca Raton, FL, p. 27.2 8-15. (Published).
  • UTAO+0371 Suwa, Y., Norton, J. M., Bollmann, A., Klotz, M. G., Stein, L. Y., Laanbroek, H. J., & Arp, D. J., (2011). Genome Sequence of Nitrosomonas sp. Strain AL212, an Ammonia-Oxidizing Bacterium Sensitive to High Levels of Ammonia.: Journal of Bacteriology/American Society Microbiology, 193: 5047-5048. (Published).
  • UTAO+371 Norton, J. M., & Stark, J. M., 2011. Methods in Enzymology Regulation and measurement of nitrification in terrestrial systems. Elsevier, Methods in Enzymology, p. 344-368. (Published).
  • UTAO+0371 Campbell, M. A., Chain, P. S.G., Dang, H., El-Sheikh, A. F., Norton, J. M., Ward, N. L., Ward, B. B., & Klotz, M. G., (2011). Nitrosococcus watsonii sp. nov., a new species of marine obligate ammonia-oxidizing bacteria that is not omnipresent in the world's oceans.Calls to validate the names "Nitrosococcus halophilus" and "Nitrosomonas mobilis.": FEMS Microbiology Ecology, 76(1): 39-48. (Published).


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Objective 1. Functional genomics of nitrifying bacteria. The genomic sequence for Nitrosomonas sp. IS79A from Dr. Annette Bollmann (Univ. of Miami) was obtained in collaboration with the Joint Genome Institute (JGI) and data analysis is ongoing. Analysis results for Nitrosomonas sp. AL212 and IS79 were presented at the meeting of the Nitrification Research Coordination Network steering committee. Genomic DNA preparation for Nitrosospira briensis and for Nitrosomonas cryotolerans is progressing. Objective 3. Nitrification ecology. Genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea have been amplified from a rangeland site in Colorado. These have been sequenced and analyzed with results presented at the International Society for Microbial Ecology and at Soil Science Society America. Real-time PCR targeting genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea have been developed and have been completed for both rangeland and agricultural soils. Book chapter reviewing the distribution and diversity of ammonia oxidizing bacteria in press to be published 2010. Book chapter on nitrogen mineralization and immobilization turnover in press to be published 2011. A website for the Nitrification Network has been established and maintained (see http://nitrificationnetwork.org/index.php). PARTICIPANTS: John Stark, Dept. of Biology, USU; Research Coordination Network for Nitrification (Nitrification Network), City of Logan Environmental Division; Standard Methods for Water and Wastewater AWWA. TARGET AUDIENCES: Scientists, educators and government agencies interested in nitrification and management of nitrogen cycling. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Improved understanding of nitrifying bacteria and archaea in soils and in wastewater systems may suggest management options for particular environments. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the limiting factors for nitrification in water delivery and wastewater treatment systems will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region. Shifts in the balance between ammonia oxidizing bacteria and ammonia oxidizing archea suggest that these organisms have distinct niches with the ammonia oxidizing archaea favored by undisturbed and low nitrogen conditions in general.

Publications

  • Norton, J. 2010. Diversity and environmental distribution of ammonia-oxidizing bacteria. ASM Press, Washington D.C., 39-55.
  • Koper, T., Habteselassie, M., Stark, J., Norton, J. 2010. Nitrification exhibits Haldane kinetics in an agricultural soil treated with ammonium sulfate or dairy waste compost.. FEMS Microbial Ecology, Federation of European Microbiological Societies, Blackwell Publishing Ltd, Oxford, England 74:316-322. DOI:10.1111/j.1574-6941.2010.00960.x.
  • Xu, L., Teng, Y., Li, Z., Norton, J., Luo, Y. 2010. Enhanced removal of polychlorinated biphenyls from alfalfa rhizosphere soil in a field study: the impact of a rhizobial inoculum. Science of the Total Environment, Elsevier B.D., 408:5, 1007-1013. 10.1016/j.scitotenv.2009.11.031.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Objective 1. Functional genomics of nitrifying bacteria. The genomic sequence for Nitrosomonas spp. AL212 from Dr. Yuichi Suwa (Japan) was obtained in collaboration with the Joint Genome Institute (JGI) and data analysis is ongoing. The genomic DNA for strain Nitrosomonas sp. IS79 has been submitted once and the request for additional DNA from the JGI will be fulfilled by collaborator Dr. Annette Bollmann (University of Miami). Analysis results for Nitrosomonas sp. AL212 were presented at the American Society for Microbiology. An international meeting on nitrification (First International Conference of Nitrification ICON-1) was planned, organized and held during July 2009 in Louisville KY. Two presentations from this project were included at the conference. Objective 2. Characterization of N cycling in a wastewater treatment system using artificial wetlands. We amplified novel sequences of ammonia oxidizing archaea from the wastewater. This is a new group of ammonia oxidiziers that we have not previously studied. The Nitrification section of Standard Methods for the Examination of Waters and Wastewaters is being revised to include molecular techniques. Objective 3. Nitrification ecology. Genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea have been amplified from a rangeland site in Colorado. These are currently being sequenced. Real-time PCR targeting genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea has been developed and is in process for both rangeland and agricultural soils. Book chapter reviewing the distribution and diversity of ammonia oxidizing bacteria accepted with revisions. Book chapter on nitrogen mineralization and immobilization turnover has been completed and revised to be published 2011. A website for the Nitrification Network has been established and maintained (see http://nitrificationnetwork.org/index.php). More than 80 scientists gathered July 5-10, 2009 at the First International Conference on Nitrification (ICoN1) at the University of Louisville, USA. The ICoN1 conference brought together investigators and students at all levels to discuss the most recent concepts in nitrification research, including the ecology, physiology, biochemistry, molecular genetics, genomics, metagenomics, transcriptomics and evolution of ammonia- and nitrite-oxidizing bacteria and archaea, and their roles in the nitrogen cycle. Participants from a wide range of disciplines from both academic, industrial and policy institutions from all over the world were present. PARTICIPANTS: John Stark, Dept. of Biology, USU; Research Coordination Network for Nitrification (Nitrification Network), City of Logan Environmental Division; Standard Methods for Water and Wastewater AWWA TARGET AUDIENCES: Scientists, educators and government agencies interested in nitrification and management of nitrogen cycling. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Improved understanding of nitrifying bacteria and archaea in soils and in wastewater systems may suggest management options for particular environments. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the limiting factors for nitrification in water delivery and wastewater treatment systems will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.

Publications

  • No publications reported this period


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Objective 1. Functional genomics of nitrifying bacteria. Revisions and further interpretation of the genome sequence for Nitrosospira multiformis were completed and the paper published. Due to difficulty with obtaining sufficient DNA from Nitrosomonas oligotropha we devised an alternative strategy and have replaced N. oligotropha with a related Nitrosomonas strain AL212 from Dr. Yuichi Suwa (Japan). The draft sequence for this organism has now been obtained and preliminary data analysis has begun. The genomic DNA for strain Nitrosomonas sp. IS79 has been submitted to Joint Genome Institute and this strain is being sequenced currently. Meetings on genome comparisons were held and further efforts in this area will be finalized before international meeting on nitrification planned for July 2009. Objective 2. Characterization of N cycling in a wastewater treatment system using artificial wetlands. Further nitrification potentials, isolation of AOB and NOB and molecular characterization of nitrifiers from the wastewater treatment lagoons and wetland soils (Logan, UT) have been accomplished. We also amplified novel sequences of ammonia oxidizing archaea in the wastewater. This is a new group of ammonia oxidiziers that we have not previously studied. Efforts to enhance nitrification earlier in the lagoon system will be crucial in promoting coupled nitrification-denitrification and minimizing nitrogen release to surface waters. Current progress in the lagoon wetland system must broaden the focus to include phosphorus cycling. A revision to the standard methods section on nitrifying bacteria was prepared by the joint task force. Objective 3. Nitrification ecology. Drafts and revisions of results from agricultural studies were prepared and are in internal review. Genes for the ammonia monooxygenase enzyme for both ammonia oxidizing bacteria and archaea have been amplified from a rangeland site in Colorado. These are in preparation for sequencing. Book chapter reviewing the distribution and diversity of ammonia oxidizing bacteria in prepapration. PARTICIPANTS: John Stark Dept. of Biology USU; Eric Davidson, City of Logan Environmental Division; Standard Methods for Water and Wastewater AWWA TARGET AUDIENCES: Scientists, educators and government agencies interested in nitrification and management of nitrogen PROJECT MODIFICATIONS: 1. Objective 1. Nitrosomonas oligotropha was replaced with Nitrosomonas sp. AL212 for genomic sequencing. We are currently analyzing draft sequence data.

Impacts
Improved understanding of nitrifying bacteria and archaea in soils and in wastewater systems may suggest management options for particular environments. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the limiting factors for nitrification in water deleivery and wastewater treatment systems will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.

Publications

  • Norton, J. 2008. Nitrification in Agricultural Soils. Nitrogen in Agricultural Systems, Agronomy Monograph 49:173-199.
  • Norton J.M., M. Klotz, L. Stein, D. Arp, P. Bottomley, P.Chain, L.J. Hauser, M.L. Land, F.W. Larimer, M. Shin, S. Starkenburg 2008. Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment. Applied and Environmental Microbiology 74:3559-3572.
  • Henry, A., W. Doucette, J. Norton, and B. Bugbee 2007. Changes in crested wheatgrass root exudation caused by flood, drought, and nutrient stress. J. Environmental Quality 36:904-912.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Objective 1. Functional genomics of nitrifying bacteria. The annotation intepretations and first publications for the genome sequences of Nitrosomonas eutropha and Nitrosospira multiformis were completed. These additional sequences have allowed us to begin comparative genome analysis. Distinguishing features of the soil ammonia oxidizer N. multiformis compared to N. europaea include: the presence of gene clusters encoding urease and hydrogenase and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a siderophore and an autoinducer were unique to N. multiformis. Gene clusters associated with outer membrane and cell envelope functions including transporters, porins; exopolysaccharide synthesis, capsule formation and protein sorting/export were abundant. Numerous sensory transduction and response regulator gene systems directed towards sensing of the extracellular environment were described. Gene clusters for glycogen, polyphosphate and cyanophycin storage and utilization provide mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments. Objective 2. Characterization of N cycling in a wastewater treatment system using artificial wetlands. Further nitrification potentials, isolation of AOB and NOB and molecular characterization of nitrifiers from the wastewater treatment lagoons and wetland soils (Logan, UT) have been accomplished. We found that lagoon water samples had potential nitrification rates near or below detection limits, too low for effective treatment of ammonia in lagoons even considering the long retention times. Several factors contribute to the weak nitrifying activity in the lagoons. These include 1) low oxygen availability 2) low surface area available for attached growth of nitrifying bacteria 3) fluctuating and transiently low ammonium availability causing competition for ammonium and 4) high and fluctuating pH. We observed that low ammonium levels in lagoon cells are not due primarily from N loss but rather N is being transferred from inorganic to organic forms. We believe that available ammonium is depleted in the primary cells by the assimilation and growth of algae and heterotrophic bacteria. This starves the nitrifiers early in the process and they do not become reestablished in the lagoon system. Soil samples from the wetland root-zones showed evidence of coupled nitrification and denitrification but these processes are spatially localized and may not be sufficient to reduce overall N contents. Denitrification and nitrification enzyme assays indicated that the root-zones of hard bulrushes have higher rates of N transformations overall. Efforts to enhance nitrification earlier in the lagoon system will be crucial in promoting coupled nitrification-denitrification and minimizing nitrogen release to surface waters. Objective 3. Nitrification ecology. Results from projects on nitrification in agricultural soils treated with dairy-wastes are summarized in Western Nutrient Management Conference Proceedings. PARTICIPANTS: John Stark Dept. of Biology USU; Eric Davidson, City of Logan Environmental Division; Standard Methods for Water and Wastewater AWWA TARGET AUDIENCES: Scientists, educators and government agencies interested in nitrification and management of nitrogen PROJECT MODIFICATIONS: Objective 3. Nitrification ecology in wildlands and in agricultural soils. Additional projects will be initiated including characterization of nitrifiers in field sites in Colorado. New graduate students funded through USDA/NRI project (Stability of Weedy Plant Communities in Western Rangelands P.I. J.M. Stark and Co P.I. J.M. Norton) will be trained and research initiated.

Impacts
Improved understanding of nitrifying bacteria in soils and their distinctive features compared to waste system nitrifying bacteria may suggest management options for particular environments. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the limiting factors for nitrification in wastewater treatment systems and agricultural soils will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.

Publications

  • Stein, L.Y., D.J. Arp, P.M. Berube, P.S.G. Chain, L. Hauser, M.S.M. Jetten, M. G. Klotz, F.W. Larimer, J. M. Norton, et al. 2007. Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha C91:implications for niche adaptation. Environmental Microbiology 9:2993-3007.
  • Norton, J.M. Habteselassie, M. Y., J. M. Stark, B. E. Miller. 2007. Manure treatments change nitrogen cycling in soils receiving repeated applications of dairy-wastes. Western Nutrient Management Conference Proceedings 7:42-44.


Progress 01/01/06 to 12/31/06

Outputs
Objective 1. Functional genomics of nitrifying bacteria. The complete genomes of the nitrifying bacteria: Nitrosospira multiformis, Nitrosococcus oceani, Nitrobacter winogradski and Nitrobacter hamburgensis have been sequenced by the Joint Genome Institute of the DOE. These data are available on their Integrated Microbial Genomes website (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) and in the GenBank Database NCBI. The Nitrobacter genome sequences are the first for the nitrite oxidizing bacteria. These sequences have allowed us to begin comparative genome analysis. Objective 2. Characterization of N cycling in a wastewater treatment system using artificial wetlands. Further nitrification potentials, isolation of AOB and NOB and molecular characterization of nitrifiers from the wastewater treatment lagoons and wetland soils (Logan, UT) have been accomplished. Nitrification potentials and populations remain low in the lagoon water. Increasing the availability of surfaces for attachment of the nitrifiers may be an option to encourage their growth and activity in the lagoons. Nitrification potentials in wetland soils were considerably higher but mass transfer limitation of oxygen and ammonium into the soils is expected to slow conversion. Nitrification potentials were consistently high in the root-zone of bulrush. Management options to promote nitrification include: 1) improved oxygen availability and surfaces for attachment in the lagoons to promote earlier nitrification and 2) cycling water levels in the wetlands to control oxygen availability and promote coupled nitrification/denitrification. Management of internal recycling between organic and inorganic N in the lagoons will be crucial for coupling nitrification to denitrification and N removal. Objective 3. Nitrification ecology. Results from projects on nitrification in agricultural soils were published. In silage corn long-term studies we found that peak plant nitrogen demand can easily be met by dairy-waste compost, but continued nitrogen release after corn harvest makes nitrate leaching likely. We suggest that careful management of compost needs to account for the amount and timing of N release from multiple year applications. We also found soil accumulation of available phosphorus and potassium was problematic. While we tend to think that applications of composted manure will be more environmentally friendly than liquid wastes; we observed that if compost is applied as the sole nitrogen source at the rates necessary for high yield there is significant risk of nitrate leaching. On the positive side, the build up of soil organic carbon was significant in the high rate compost treated plots, approximately doubling the soil organic carbon storage. The management of dairy wastes requires a careful consideration of the timing of carbon versus nitrogen availability to the soil microorganisms. Additions of high carbon materials or use of a cover crop to trap available nitrogen after corn harvest are possible solutions to prevent leaching.

Impacts
Livestock and crop producers are looking for ways to effectively use the nutrient content of manures. Livestock producers need to ensure that the nutrients are not causing harm to the environment and crop producers look to organic waste materials in lieu of inorganic fertilizers or as a primary component of an organic production system. Crop and livestock producers benefit from a better understanding of the factors influencing nitrogen release and movement from dairy-waste materials when applied to the soil. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the limiting factors for nitrification in wastewater treatment systems will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.

Publications

  • Habteselassie, M. Y., J. M. Stark, B. E. Miller, S. G. Thacker and J. M. Norton. 2006. Gross nitrogen transformations in an agricultural soil after repeated dairy-waste application. Soil Sci. Soc. Am. J. 70: 1338-1348.
  • Habteselassie, M. Y., J. M. Stark, B. E. Miller, S. G. Thacker and J. M. Norton. 2006. Soil nitrogen and nutrient dynamics after repeated application of treated dairy-waste. Soil Sci. Soc. Am. J. 70:1328-1337.
  • Klotz, M.G., D. J. Arp, P. S.G. Chain, A. F. El-Sheikh, L. J.Hauser, N. G. Hommes, F. W. Larimer, S. A. Malfatti, J. M. Norton, A. T. Poret-Peterson, L. M. Vergez, and B. B. Ward. 2006. The complete genome sequence of the marine, chemolithoautotrophic, ammonia- oxidizing bacterium Nitrosococcus oceani ATCC19707. Appl. Environ. Microbiol. 72: 6299-6315.
  • Henry, A., W. Doucette, J. Norton, S. Jones, J. Chard and B. Bugbee. 2006. An axenic culture system for optimal growth in long-term studies. J. Environmental Quality 35:590-598.
  • Henry, A., W. Doucette, J. Norton, and B. Bugbee. 200X. Changes in crested wheatgrass root exudation caused by flood, drought, and nutrient stress. J. Environmental Quality. in press


Progress 01/01/05 to 12/31/05

Outputs
Objective 1. Functional genomics of nitrifying bacteria. The complete genomes of the nitrifying bacteria: Nitrosospira multiformis, Nitrosococcus oceani, and Nitrobacter winogradski have been sequenced by the Joint Genome Institute of the Department of Energy. These data are available on their Integrated Microbial Genomes website (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) and in the GenBank Database of the National Center for Biotechnology Information. The genomes of the nitrifying bacteria: Nitrosomonas eutropha and Nitrobacter hamburgensis are in the draft stage and are expected to be completed in the next year. The genome sequences of Nitrobacter winogradsky and Nitrobacter hamburgensis are the first for the nitrite oxidizing bacteria. These sequences have allowed us to begin comparative genome analysis. Some of the observations include: 1) multiple ammonia monooxygenase operons in Nitrosospira spp. may add to flexibility under fluctuating ammonia availability such as found in soils, 2) urease encoding genes have been lost from N. europaea and from N. eutropha; this function holds little advantage for these organisms that are adapted to high ammonia availability and moderate pH, 3) there are many novel multi-copper oxidases in nitrifying bacteria; a role in the reduction of nitrite toxicity for these is suggested for N. europaea and N. eutropha but more functional genomics characterization is needed for the other bacteria, and 4) genes involved in sucrose metabolisms were identified and likely function in osmotic and matric stress management or in energy / C storage. Objective 2. Characterization of N cycling in a wastewater treatment system using artificial wetlands. Further nitrification potentials, isolation of AOB and NOB and molecular characterization of nitrifiers from the wastewater treatment lagoons and wetland soils (Logan, UT) have been accomplished. The AOB identified are of the Nitrosomonas lineage. Nitrification potentials and populations are too low in the lagoon water to be effective in converting ammonium to meet regulatory limits. Nitrification potentials in wetland soils were considerably higher but mass transfer limitation into the soils is expected to slow conversion. Management options to promote nitrification include improved oxygen availability in the lagoons to promote earlier nitrification. Increasing the availability of surfaces for attachment of the nitrifiers may be an option to encourage their growth and activity in the lagoons. Management of internal recycling between organic and inorganic N in the lagoons will be crucial for coupling nitrification to denitrification and N removal. Objective 3. Nitrification ecology. Projects on nitrification in agricultural soils were completed and two graduate students have finished their degrees. Site visit to the Ogden Canyon poplar site and a framework for collaboration with the research group of Prof. Stephen Hart of Northern Arizona University has been established. Characterization of the ammonia oxidizing bacteria at this site is ongoing.

Impacts
The genome sequence of Nitrosospira multiformis has been analyzed and this information offers insight into the function of one of the most common soil nitrifiers in agricultural systems. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the factors limiting nitrification rates and the role of aeration in wastewater lagoons will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.

Publications

  • Habteselasssie, M. Y., 2005. Shifts in ammonia-oxidizing bacteria community structure and nitrogen transformations with dairy waste application in an agricultural soil. Ph.D. diss. Utah State University, Logan, USA.
  • Koper T.E. 2006. Functional diversity in autotrophic ammonia-oxidizing bacteria from agricultural soils. M.Sc. thesis Utah State University, Logan, USA.
  • Norton J.M, M.G. Klotz, L.Y. Stein, P.J. Bottomley and D.J. Arp. 2005. Nitrifier genomics: links between habitats and functional gene diversity. ASA-CSSA-SSSA Abstracts. Salt Lake City, UT.
  • Koper, T. E., J.M. Stark, M.Y. Habteselassie, J.M. Norton. 2005. Nitrification exhibits haldane kinetics in an agricultural soil treated with ammonium and dairy waste. ASA-CSSA-SSSA Abstracts. Salt Lake City, UT.
  • Habteselassie, M.Y, J.M. Stark, B.E. Miller, S.M. Thacker, J.M. Norton. 2005. Use of real-time and conventional PCR to quantify and profile ammonia-oxidizing bacteria in dairy waste treated agricultural soil. ASA-CSSA-SSSA Abstracts. Salt Lake City, UT.
  • Norton J.M, L.Y. Stein M.G. Klotz, P. S. G. Chain, F.W. Larimer, M. Land and D.J. Arp. 2005. The genome of Nitrosospira multiformis: functional and evolutionary insights from a comparison with Nitrosomonas europaea. ASM Abstracts. Atlanta, GA.


Progress 01/01/04 to 12/31/04

Outputs
Objective 1. Functional genomics of nitrifying bacteria. A project to extend genomic data to a selection of nitrifying bacteria (Nitrosomonas eutropha, Nitrosospira multiformis, Nitrosococcus oceani, Nitrobacter winogradski and Nitrobacter hamburgensis) was proposed during 2003 by a collaborative group of scientists interested in both ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) The Joint Genome Institute (DOE) has now accomplished draft genome sequences of several of these AOB and NOB; these are being used for comparative genome analysis. The genome sequences of Nitrobacter winogradsky and N. hamburgensis are the first for NOB. An all collaborator meeting is scheduled for February 2005. The curation and analysis of the N. multiformis genome sequence is ongoing. We are particularly interested in comparative genomics, examining genes and operons encoding key metabolic functions in the AOB, and the evolutionary implications of protein/gene phylogenies. Ammonia monooxygenase and hydroxylamine oxidoreductase encoding genes are highly conserved and congruent with 16S rRNA in all AOB as was expected from known functional and evolutionary relationships. Urease encoding genes have been lost from N. europaea but a urea transporter gene was found. This gene was closely related to the UT encoding genes in nitrosospiras that have intact ure operons and are known to be ureolytic. Surprisingly, the genes encoding RUBP-carboxylase of N. europaea are distinctly divergent from those of other AOB including N. multiformis. Insertion and repetitive sequences are numerous in the genomes; highlighting the importance of genome rearrangements and possible lateral gene transfers in the evolution of functional diversity in the AOB. Objective 2. Characterization of N cycling in a wastewater treatment system using artificial wetlands. A culture of Nitrosomonas oligotropha has been received from Germany and is now maintained in the Norton lab. This strain is often found in wastewater treatment systems. Nitrification potentials, isolation of AOB and NOB and molecular characterization of nitrifiers from the wastewater treatment lagoons and wetland soils (Logan, UT) have been accomplished. The AOB identified are of the Nitrosomonas lineage. Nitrification potentials and populations are too low in the lagoon water to be effective in converting ammonium to meet regulatory limits. Nitrification potentials in wetland soils were considerably higher but mass transfer limitation into the soils is expected slow conversion. Increasing the availability of surfaces for attachment of the nitrifiers is an important to encourage their growth and activity in the lagoons. Management options to promote nitrification are being explored. Objective 3. Nitrification ecology in wildlands. Planning meetings and collaborative arrangements have been initiated and additional funding opportunities are being explored.

Impacts
The genome sequence of Nitrosospira multiformis has been completed by the Joint Genome Institute and this information offers insight into the function of one of the most common soil nitrifiers. The management of nitrification is of importance in protecting ground and surface waters from nitrate pollution, a major cause of eutrophication. Improved understanding of the limiting factors for nitrification in wastewater treatment systems will help municipal and other government entities in preventing water pollution and planning for water reuse in the semi-arid and arid Intermountain West region.

Publications

  • Norton J.M, M.G. Klotz and D.J. Arp. 2004. Functional genomics of autotrophic ammonia oxidizing bacteria. International Society of Microbial Ecology Abstracts.