Source: UNIVERSITY OF ILLINOIS submitted to
LEVERAGING PLANT-MICROBE INTERACTIONS TO ENHANCE SUSTAINABILITY OF BIOFUEL CROPS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
0205664
Grant No.
(N/A)
Project No.
ILLU-875-374
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2011
Project End Date
Sep 30, 2016
Grant Year
(N/A)
Project Director
Kent, A. D.
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
Natural Resources & Environmental Sciences
Non Technical Summary
Plant productivity is often limited by availability of nitrogen, and application of nitrogen fertilizer can account for a major portion of fossil fuel use in agricultural systems. Because nitrogen fertilizers are primarily derived from fossil-fuel intensive processes, low N fertilizer inputs are especially important for the sustainability of bioenergy crops, such as Miscanthus. Nitrogen-fixing bacteria have demonstrated the capacity to supply significant levels of biologically fixed nitrogen in some non-leguminous crops; however, we currently lack information on the factors that govern the abundance and activity of these beneficial microbes in perennial grasses. There is a critical need to better understand interactions among associative diazotrophs and their plant hosts in order to promote these relationships to improve plant nutrition. Our objective in this research is to quantify the contribution of nitrogen-fixing bacteria to Miscanthus plant N, and to identify plant and microbial traits and environmental factors that influence diazotroph colonization and activity. Previous work in natural and agricultural systems indicates that plant genetic variability affects the identity, abundance, and activity of bacterial endophytes, including associative N-fixers, and thus that enhancing beneficial microbial interactions are a promising approach for plant breeding programs. Our working hypothesis is that understanding factors that influence plant-microbe mutualisms will allow for the optimization of associative N fixation in perennial grasses, and thereby enhance sustainability of these bioenergy crops. We have proposed greenhouse and field trials to survey the range of plant-microbe associations in Miscanthus species, which is synergistic with an extensive Miscanthus breeding program at UIUC.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110107015%
1021629107070%
2021629107015%
Goals / Objectives
Our objective in this research is to quantify the contribution of nitrogen-fixing bacteria to Miscanthus plant N, and to identify plant and microbial traits and environmental factors that influence diazotroph colonization and activity. Understanding factors that influence plant-microbe mutualisms will allow for the optimization of associative N fixation in perennial grasses, and thereby enhance sustainability of these bioenergy crops. We hypothesize that the efficiency of plant-microbe mutualisms will vary as a function of microbial community structure and environmental conditions. We further hypothesize that the efficiency of such mutualisms varies among plant species or genotypes. Nitrogen efficiency is important for the sustainability of crops, and especially critical for biofuel feedstocks. Improving our understanding of the biological mechanisms governing beneficial associations between gramineous bioenergy crops and nitrogen-fixing bacteria has potential to enhance the sustainability of biofuel feedstocks. We are currently in the early stages of breeding perennial grasses for use as bioenergy feedstocks, making this a key time to evaluate the sustainability and N efficiency of these crops. There is relatively little information on how plant genetic variation affects diazotroph associations and natural N fixation. However, our preliminary data indicates that plant genetics play an important role. Thus, the results of this study are expected to facilitate efforts to breed Miscanthus for improved efficiency of plant-microbe interactions.
Project Methods
We will estimate the direct and interactive effects of plant genotype and soil environment on the diversity, abundance, and identity of nitrogen-fixing bacteria associated with Miscanthus using a factorial experimental design carried out in a greenhouse. Plant yield and N content will be assessed and compared with molecular analyses of plant associated microbial communities. We will use the outcome of the greenhouse experiment to select cultivars that we will use to assess the contribution of plant-diazotroph mutualisms to N-fixation and plant growth across Miscanthus genotypes in response to changing soil types and nitrogen availability. For this field study, we will employ an isotope dilution model approach to evaluate N fixation, and we will use molecular methods targeting nitrogen-fixing bacteria to compare diazotroph assemblages associated with each Miscanthus population. Diazotroph abundance will be assessed in rhizome (endophyte) and rhizosphere soil bacterial communities using a quantitative PCR assay targeting the nifH gene. We will also compare shifts in the diversity and composition of diazotroph assemblages over time using nifH terminal restriction length polymorphism.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One graduate student has been trained, and a postdoctoral researcher is also starting to participate in this project. The graduate student and PI have expanded their capabilities in bioinformatics. How have the results been disseminated to communities of interest? Conference presentations and publications. What do you plan to do during the next reporting period to accomplish the goals? We will continue to analyze the results from a large sequencing project (recently completed) and evaluate the Miscanthus associated microbiome as a function of plant genotype. As samples from our large N-15 isotope dilution experiment are processed, we will be able to quantify N fixation in mature Miscanthus.

Impacts
What was accomplished under these goals? Identification of diazotrophs:To fulfill our objective of quantifying the contribution of nitrogen-fixing bacteria to Miscanthus plant N, we conducted a field experiment on mature Miscanthus plant during its growing season (May - October) using N-15 isotope dilution approach. A total of 10 sample collecting time points were made for five replicated fields during the experimental period. Total plant available nitrogen, N-15 labeled plant available nitrogen in bulk soil, and the accumulation of N-15 labeled nitrogen in plant tissue were monitored during the experiment. Nitrogen-fixing bacteria community from rhizosphere and endosphere of Miscanthus plant were also collected at each sampling time. So far, we finished measuring the total and N-15 labeled plant available nitrogen in bulk soil. The rest of the samples are still under processing. Identifying important ecological drivers:Identifying the environmental factors:Identical M. × giganteus rhizomes cultivated in four field sites in NE, KY, IL, and NJ were collected for microbial analysis after 3 years of cultivation. Our data indicated that local soil edaphic factors had great influence to the diazotroph assemblage in rhizosphere, and less influence to the endosphere community. Despite differences in regional microbial species pools, the same Miscanthus genotype selects for specific assemblages of endophytic diazotrophs. Surprisingly, although the total soil nitrogen affected the richness and diversity of the diazotrophs in endosphere to some extent, the microbial assemblage seemed to receive little influence from the artificial nitrogen fertilization. Our 20-week greenhouse experiment using four different Miscanthus genotypes indicated that different Miscanthus genotypes tend to recruit different diazotroph species from the bulk soil. Using 454-generated nifH sequences, we are able to identify the main diazotroph species that reside inside the plant tissue and rhizosphere soil, and also we identified OTUs that respond to different Miscanthus genotypes.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Keymer, D. P. and A. D. Kent. Contribution of nitrogen fixation to first year Miscanthus x giganteus. GCB Bioenergy, in press. doi: 10.1111/gcbb.12095.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Kent, A.D. Assessing the Miscanthus-associated microbiome: Prospects for improving sustainability. Invited presentation for the Cluster of Excellence in Plant Sciences, University of Cologne, Germany. September 6, 2013.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Kent, A.D. Micro-managing sustainability: Ecology of diazotrophs associated with Miscanthus. Invited presentation for the 2nd Workshop on Genetics of Maize-Microbe Interactions. Donald Danforth Plant Science Center, Saint Louis, MO. February 24-27, 2013.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Li, D., T. Voigt and A.D. Kent. Plant and soil effects on bacterial communities associated with Miscanthus � giganteus rhizosphere and rhizomes. Poster presentation at the 98th Annual Meeting of the Ecological Society of America, Minneapolis, MN. August 4-9, 2013.


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

Outputs
OUTPUTS: Activities: Participation in the JGI Community Sequencing Project focused on plant-associated microbiomes. Events: Kent, A.D. Micro-managing sustainability: Ecology of diazotrophs associated with Miscanthus. Invited presentation for the 13th Symposium on Nitrogen Fixation with Non-Legumes, Munich, Germany. September 6-7, 2012. Kent, A.D. Micro-managing sustainability: Ecology of the Miscanthus-associated microbiome. Invited presentation for the 28th New Phytologist Symposium:
Functions and ecology of the plant microbiome.
Rhodes, Greece, 18-21 May, 2012. Kent, A.D. Micro-managing sustainability: Ecology of diazotrophic bacteria associated with perennial grasses. Invited presentation for the School of Biological Sciences, Illinois State University. Bloomington, IL. March 1, 2012. Kent, A.D. Micro-managing sustainability: Ecology of the Miscanthus-associated microbiome. Oral presentation at the 4th Annual Argonne Soil Metagenomics Meeting, Chicago, IL. October 3-5, 2012. Li, D. and A.D. Kent. Ecology of diazotrophs associated with Miscanthus. Poster presentation at the 28th New Phytologist Symposium:
Functions and ecology of the plant microbiome.
Rhodes, Greece, May 18-21, 2012. PARTICIPANTS: Angela Kent - PI and Dongfang Li - Graduate student who analyzes microbial communities associated with Miscanthus Joint Genome Institute - provided microbiome sequencing. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our preliminary data in Miscanthus indicate that the N efficiency of M. x giganteus 'Illinois' is derived, in part, from associative N fixation. These conclusions stem from modeling as well as documentation of diazotroph presence and activity in Miscanthus roots, complemented by a field-scale isotope dilution experiment demonstrating 16% of N acquired by first-year plants via biological N fixation. In 2012, we conducted a follow up isotope dilution study to evaluate N fixation in replicated mature Miscanthus plots. Soils were labeled with dual-labeled ammonium nitrate at the start of the growing season. Five replicate samples were collected at each of 10 time points from May - October. These samples are currently under evaluation for soil N, plant N and isotopic ratios, as well as composition and abundance of diazotrophs. Four distinct Miscanthus genotypes were grown in a greenhouse experiment in soils from two locations known to differ in N levels and diazotroph abundance. Endophytic diazotrophs recruited by each plant genotype were distinct from the diazotroph assemblages in the rhizosphere (which were similar to the diazotroph assemblages in the bulk soil). Distinct diazotroph assemblages were recruited by diploid Miscanthus sinensis and M. sacchariflorus. Triploid and tetraploid Miscanthus x giganteus (hybrids of M. sinensis and M. sacchariflorus) had diazotroph assemblages that were intermediate in composition compared to both parents. Diazotroph abundance was compared among Miscanthus x giganteus plots with 3 different N levels replicated in 4 states. Diazotroph abundance and composition varied among states, but not as a function of N fertilization. 1,700 Miscanthus-associated microbial samples from 20 different native populations, 30 different naturalized populations, and 7 agricultural sites were submitted to the JGI Community Sequencing Project for microbiome analyses based on the 16S rRNA genes. Sequencing was recently completed, and analysis of the results is underway. Plant genotyping of samples from all locations is currently underway to enable evaluation of plant genotype controls on the Miscanthus-associated microbiome. DNA sequencing of nifH will be carried out for representative samples (based on nifH T-RFLP).

Publications

  • No publications reported this period


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

Outputs
OUTPUTS: Soil microorganisms are responsible for removal of nitrate from the terrestrial ecosystem and reducing pollution of surface waters. Understanding the response of microbial communities and processes to land use change is essential for restoring wetland water quality functions. This research will help to improve wetland assessment protocols to include below-ground processes that affect water quality. We are using the data collected in this research to generate models of soil chemical and physical variables and plant community variables that influence denitrification data. Results of our wetland microbial ecology investigations may also aid in development of management prescriptions to enhance the nutrient-removal function of constructed and restored wetlands in Illinois. Outputs include recommendations for soil parameters that could be included in assessment and monitoring approaches. We have improved our capacity to predict the effect of land use changes on microbially-mediated processes. We investigated microbial community structure and denitrification function in restored floodplains throughout Illinois, Michigan, and in a floodplain about to undergo restoration in Pennsylvania. In all cases, microbial N cycling processes (nitrification, denitrification) were measured, and DNA was extracted for characterization of microbial communities. Results were presented to wetland professionals and researchers at regional and national meetings: Peralta, A.L., J.W. Matthews, Sarah Ludmer, Eric Johnston and A.D. Kent. Assessment of abiotic controls on microbial community structure and function: Linking microbial community structure and water quality function in restored floodplain wetlands, and Linking microbial community structure to water quality function: Investigating nitrogen cycling influenced by hydrology in wetland ecosystems. Presented at the Midwest-Great Lakes Chapter Society for Ecological Restoration, Lewiston, IL April 1-2, 2011. One PhD thesis and one MS thesis also resulted. We completed investigations of pre- and post-restoration microbial communities in restored wetlands sites in PA with K. Forshay (U.S. EPA) to determine if buried wetland soils will regain denitrification function following restoration. We delivered a 6-week long learning module to the 8th grade environ. science class at the Campus Middle School for Girls. In addition, results of this work were included in Environmental Microbiology and Restoration Microbiology courses. We developed an inquiry based graduate level hands-on microbial ecology methods course that allowed 14 graduate students to investigate microbial community structure and function at the Emiquon wetland restoration site near Havana, IL. PARTICIPANTS: Angela Kent: PI, project management, experimental design. Ariane Peralta: technician/PhD student, conducted field work at Grundy County site in 2007, received training in soil chemistry analyses, denitrification assays, nitrification assays, DNA extraction, and molecular microbial community analyses. Additional field work was conducted in 2008 at The Nature Conservancy's Emiquon wetland restoration site near Havana, IL, to monitor wetland microbial activity and composition in relation to fluctuating water levels during the initial stages of wetland restoration. In 2009, field studies were expanded to 2 sites in Illinois (Emiquon and a restored wetland near St. Joseph, IL) and 2 sites at Kellogg Biological Station in Michigan. Results of this research have been presented at state, national, and international conferences. Jason Koval: MS student, conducting field and lab work at a restoration site in PA, in conjunction with EPA researchers. Partnerships were developed with wetland researchers at University of Illinois at Springfield, the Emiquon Field Station, Kellogg Biological Station, the Illinois State Geological Survey, the EPA, and The Nature Conservancy. Jason completed a summer internship with the EPA in Ada, OK, while investigating microbial community structure and function buried wetland sediments. Eric Johnston: Undergraduate student, conducted field work at The Nature Conservancy's Emiquon wetland restoration site near Havana, IL, to monitor wetland microbial activity and composition in relation to fluctuating water levels during the initial stages of wetland restoration. Carried out microbial analyses and analyzed data to complete an undergraduate distinction project. Sarah Ludmer: Undergraduate student, conducted field work at The Nature Conservancy's Emiquon wetland restoration site near Havana, IL, and a restored wetland in St. Joseph, IL to monitor wetland microbial activity and composition in relation to fluctuating water levels during the initial stages of wetland restoration. Working to complete an undergraduate distinction project. 14 graduate students enrolled in NRES 598 Microbial Ecology Methods investigated microbial community structure and function at the Emiquon wetland restoration site near Havana, IL. Campus Middle School for Girls: Project participants developed hands-on aquatic science activities to introduce middle-school girls to concepts in water quality and environmental science. Mentors for this activity included Kent and two female graduate students in the Kent lab to serve as positive role models for women in science. In April and May 2010, we delivered a 6-week long learning module on the urban water cycle to the 8th grade environmental science class. TARGET AUDIENCES: Wetland researchers, natural resource managers, Extension professionals, undergraduate students interested in conservation of natural resources, landowners, the public, and state and federal agencies concerned with natural resources. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Bacterial community structures differed significantly between restored and reference wetlands; denitrifier community assemblages were similar among reference sites but highly variable among restored sites. Potential denitrification was highest in the reference wetland sites. These data demonstrate that wetland restoration efforts have not successfully restored denitrification and that differences in potential denitrification rates may be due to distinct microbial assemblages observed in restored and reference (natural) wetlands. We have identified gradients in soil moisture and soil fertility that were associated with differences in microbial community structure. Microbial function was influenced by bacterial community composition and soil fertility. Our investigations of nitrogen cycling across a moisture gradient demonstrated that denitrifiers and nitrifiers have different responses to changes in moisture levels such that distinct differences in soil moisture produced distinct assemblages of nitrifiers and denitrifiers. Our results suggest that specific taxa may be restricted to particular habitats across space, and the interaction between abiotic and biotic factors at a site may drive ecosystem function in predictable ways. In our investigation of wetland soils to be remediated by removal of legacy (dam-deposited) sediments in PA, we demonstrated that the buried wetland soils have significantly lower denitrification rates compared to natural reference wetlands, and this difference in denitrification rate is also correlated with differences in denitrifier assemblages. This observation calls into question the prospects for successful restoration of denitrification following re-establishment of wetland hydrology, and prompted a transplant experiment, where buried wetland soils were transplanted to a natural wetland to experience wetland hydrology. Shifts in microbial community structure and function resulting from changes in hydrology will be monitored. Denitrification function increased somewhat, but was not fully restored after microbial communities from the soil targeted for restoration were exposed to wetland conditions for three months.

Publications

  • Rigonato, J., Alvarenga, D.O., Andreote, F.D., Dias, A.C.F., Melo, I.S., Kent, A.D. and Fiore, M.F. 2011. Cyanobacterial diversity in the phyllosphere of a mangrove forest. FEMS Microbiol. Ecol. (In Press).
  • Peralta, A.L. 2011. Soil microbial community structure and function along environemtnal gradients: Implications for wetland nitrogen cycling. PhD. Dissertation, Program in Ecology, Evolution and Conservation Biology, UIUC.
  • Koval, J. 2011. Assessing restoration potential in relict wetland soils: Investigarting the effect of wetland hydrology on soil microbial community composition and denitrification potential. M.S. Thesis, Natural Resources and Environmental Sciences, UIUC.


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

Outputs
OUTPUTS: Soil microorganisms are responsible for removal of nitrate from the terrestrial ecosystem and reducing pollution of surface waters. Understanding the response of microbial communities and processes to land use change is essential for restoring wetland water quality functions. This research will help to improve wetland assessment protocols to include below-ground processes that affect water quality. We are using the data collected in this research to generate models of soil chemical and physical variables and plant community variables that influence denitrification data. Results of our wetland microbial ecology investigations may also aid in development of management prescriptions to enhance nutrient-removal function of constructed and restored wetlands in Illinois. Outputs will include recommendations for soil parameters that could be included in assessment and monitoring approaches. We will also improve our capacity to predict the effect of land use changes on microbially-mediated processes. To accomplish our goals, time-series observations were carried out in restored floodplains in Havana and St. Joseph, Illinois during summer 2010, and this work was complemented by lab mesocosm experiments to test hypotheses arising from field observations. In all cases, microbial N cycling processes (nitrification, denitrification) were measured, and DNA was extracted for characterization of microbial communities. Results were presented to wetland professionals and researchers at regional and national meetings: Peralta. A.L., J.W. Matthews, and A.D. Kent. Linking microbial community structure to water quality function: Investigating nitrogen cycling influenced by hydrology in wetland ecosystems. Presented at the 13th International Symposium on Microbial Ecology, Seattle, WA. August 22-27, 2010, and the Illinois Water Conference, Champaign, IL. Oct. 5-7, 2010 Peralta. A.L., J.W. Matthews, and A.D. Kent. Investigating variation in microbial community composition along a moisture gradient. Emiquon Science Meeting, Lewistown, IL. March 4, 2010. In addition, this project was leveraged to establish several collaborations and additional funding. These include: an NSF-funded microbial ecology study in a restored floodplain with M. Lemke (University of Illinois at Springfield), an NSF-funded investigation of denitrifying microbes in an engineered alternative to wetlands in collaboration with L. Rodriguez and J. Zilles at UIUC, and characterization of diazotrophs in mangrove ecosystems in Brazil with colleagues at ESALQ. We also began investigations of pre- and post-restoration microbial communities in restored wetlands sites in PA with K. Forshay (U.S. EPA) to determine if buried wetland soils will regain denitrification function following restoration. Finally, we developed hands-on aquatic science activities to introduce middle-school students to concepts in water quality and environmental science. We delivered a 6-week long learning module to the 8th grade environmental science class at the Campus Middle School for Girls. In addition, results of this work were included in an Environmental Microbiology and Applied Microbial Ecology courses taught by PI Kent. PARTICIPANTS: Angela Kent: PI, project management, experimental design. Ariane Peralta: technician/PhD student, conducted field work at Grundy County site in 2007, received training in soil chemistry analyses, denitrification assays, nitrification assays, DNA extraction, and molecular microbial community analyses. Additional field work was conducted in 2008 at The Nature Conservancy's Emiquon wetland restoration site near Havana, IL, to monitor wetland microbial activity and composition in relation to fluctuating water levels during the initial stages of wetland restoration. In 2009, field studies were expanded to 2 sites in Illinois (Emiquon and a restored wetland near St. Joseph, IL) and 2 sites at Kellogg Biological Station in Michigan. Results of this research have been presented at state, national, and international conferences. Jason Koval: MS student, conducting field and lab work at a restoration site in PA, in conjunction with EPA researchers. Partnerships were developed with wetland researchers at University of Illinois at Springfield, the Emiquon Field Station, Kellogg Biological Station, the Illinois State Geological Survey, the EPA, and The Nature Conservancy. Jason completed a summer internship with the EPA in Ada, OK, while investigating microbial community structure and function buried wetland sediments. Eric Johnston: Undergraduate student, conducted field work at The Nature Conservancy's Emiquon wetland restoration site near Havana, IL, to monitor wetland microbial activity and composition in relation to fluctuating water levels during the initial stages of wetland restoration. Carried out microbial analyses and analyzed data to complete an undergraduate distinction project. Sarah Ludmer: Undergraduate student, conducted field work at The Nature Conservancy's Emiquon wetland restoration site near Havana, IL, and a restored wetland in St. Joseph, IL to monitor wetland microbial activity and composition in relation to fluctuating water levels during the initial stages of wetland restoration. Working to complete an undergraduate distinction project. Campus Middle School for Girls. Project participants developed hands-on aquatic science activities to introduce middle-school girls to concepts in water quality and environmental science. Mentors for this activity included Kent and two female graduate students in the Kent lab to serve as positive role models for women in science. In April and May 2010, we delivered a 6-week long learning module on the urban water cycle to the 8th grade environmental science class. TARGET AUDIENCES: Wetland researchers, natural resource managers, Extension professionals, undergraduate students interested in conservation of natural resources, landowners, the public, and state and federal agencies concerned with natural resources. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Bacterial community structures differed significantly between restored and reference wetlands; denitrifier community assemblages were similar among reference sites but highly variable among restored sites throughout the mitigation bank. Potential denitrification was highest in the reference wetland sites. These data demonstrate that wetland restoration efforts in this mitigation bank have not successfully restored denitrification and that differences in potential denitrification rates may be due to distinct microbial assemblages observed in restored and reference (natural) wetlands. We have identified gradients in soil moisture and soil fertility that were associated with differences in microbial community structure. Microbial function was influenced by bacterial community composition and soil fertility. Our investigations of nitrogen cycling across a moisture gradient demonstrated that denitrifiers and nitrifiers have different responses to changes in moisture levels such that distinct differences in soil moisture produced distinct assemblages of nitrifiers and denitrifiers. Our results suggest that specific taxa may be restricted to particular habitats across space, and the interaction between abiotic and biotic factors at a site may drive ecosystem function in predictable ways. In our investigation of wetland soils to be remediated by removal of legacy (dam-deposited) sediments in PA, we demonstrated that the buried wetland soils have significantly lower denitrification rates compared to natural reference wetlands, and this difference in denitrification rate is also correlated with differences in denitrifier assemblages. This observation calls into question the prospects for successful restoration of denitrification following re-establishment of wetland hydrology, and prompted a transplant experiment, where buried wetland soils were transplanted to a natural wetland to experience wetland hydrology. Shifts in microbial community structure and function resulting from changes in hydrology will be monitored.

Publications

  • Peralta, A.L., Matthews, J.W. and Kent, A.D. 2010. Microbial community structure and denitrification function in a wetland mitigation bank. Applied and Environmental Microbiology 76:4702-4215.


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

Outputs
OUTPUTS: Land use change and increased nutrient inputs in combination with wetland losses exacerbate degradation of Illinois waterways and downstream waters. Wetland restoration can potentially mitigate nutrient loading and improve water quality. Soil microorganisms are responsible for removal of nitrate from the terrestrial ecosystem and reducing pollution of surface waters. Understanding the response of microbial communities and processes to land use change is essential for restoring wetland water quality functions. Links between currently measured environmental parameters and microbial community structure and function could help develop monitoring indicators for specific ecosystem functions. This research will help to improve wetland assessment protocols to include below-ground processes that affect water quality. We are using the data collected in this research to generate models of soil chemical and physical variables and plant community variables that influence denitrification data. Results of our wetland microbial ecology investigations may also aid in development of management prescriptions to enhance nutrient-removal function of constructed and restored wetlands in Illinois. Outputs will include recommendations for soil parameters that could be included in assessment and monitoring approaches. We will also improve our capacity to predict the effect of land use changes on microbially-mediated processes. Results were presented to wetland professionals and researchers at several national meetings: Kent, A.D., A.L. Peralta, D.N. Flanagan, J.W. Matthews. A Soil Microbial Ecology Perspective of Wetland Restoration. Poster presentation at the 109th General Meeting of the American Society for Microbiology, Philadelphia, PA. May 17-21, 2009. Peralta, A.L., J.W. Matthews, A.D. Kent. Investigating Microbially-Mediated Nitrogen Cycling During Early Floodplain Development. Poster presentation at the 2009 Great Lakes Regional Biogeochemistry Conference, Kellogg Biological Station, Hickory Corners, MI. May 28-29, 2009. Peralta, A.L., J.W. Matthews, A.D. Kent. Investigating Microbially-Mediated Nitrogen Cycling During Early Floodplain Development. Poster presentation at the 94th Ecological Society of America Annual Meeting, Albuquerque, NM. August 2-7, 2009. Kent, A.D. Assessing the Effects of Land Use Change on Microbial Communities and Their Activities. Oral presentation at the 2009 Great Lakes Regional Biogeochemistry Conference, Kellogg Biological Station, Hickory Corners, MI. May 28-29, 2009. Kent, A.D. Assessing the Effects of Land Use Change on Microbial Communities and Their Activities. Oral presentation to the Illinois State Geological Survey, Champaign, IL. October 5, 2009. A M.S. thesis was also produced from this work: D.N. Flanagan. 2009. Microbial community structure and function in wetland ecosystems. Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign. Teaching materials were developed from this wetland microbial ecology work and incorporated into several courses taught by the PI: Soil Microbiology, Environmental Microbiology, and Microbial Ecology Methods. PARTICIPANTS: Angela Kent: PI, project management, experimental design. Diana Flanagan: MS student, conducted fieldwork in 2006 and 2007, received training in denitrification assays, soil chemistry analyses, DNA extraction, molecular microbial community analyses. Diana presented the results of her research at state and national conferences. Completed her M.S. degree in Natural Resources and Environmental Sciences in January, 2009. Ariane Peralta: technician/PhD student, conducted field work at Grundy County site in 2007, received training in soil chemistry analyses, denitrification assays, nitrification assays, DNA extraction, molecular microbial community analyses. Additional field work was conducted in 2008 at The Nature Conservancy's Emiquon wetland restoration site near Havana, IL, to monitor wetland microbial activity and composition in relation to fluctuating water levels during the initial stages of wetland restoration. In 2009, field studies were expanded to 2 sites in Illinois (Emiquon and a restored wetland near St. Joseph, IL) and 2 sites at Kellogg Biological Station in Michigan. Results of this research have been presented at state, national, and international conferences. Jason Koval: MS student, conducting field and lab work at a restoration site in PA, in conjunction with EPA researchers. Partnerships were developed with wetland researchers at University of Illinois at Springfield, the Emiquon Field Station, Kellogg Biological Station, the Illinois State Geological Survey, the EPA, and The Nature Conservancy. TARGET AUDIENCES: Wetland researchers, natural resource managers, extension professionals, undergraduate students interested in conservation of natural resources, landowners, public, and state and federal agencies concerned with natural resources. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our research has explored the hypothesis that current wetland restoration efforts are not effectively restoring microbial community structure and wetland ecosystem functions lost due to land use change. Soil samples collected from paired natural and constructed wetlands throughout Illinois were assayed for denitrification activity using the acetylene block method. Molecular "DNA fingerprinting" approaches were used to evaluate the composition of the soil microbial community, and to compare the microbial populations involved in denitrification using the nosZ gene (a bacterial gene involved in denitrification). Plant community and soil chemistry data were also collected. Canonical correspondence analysis was used to compare microbial assemblages among the different wetland sites, and to elucidate potential ecological drivers. Our results indicate that microbial community structure and denitrification activity differed significantly between natural and constructed wetlands. Soil nitrogen, total organic carbon, C:N, and moisture were the most significant explanatory variables. Constructed wetland sites that meet the criteria for jurisdictional wetlands (for vegetation, soil, and hydrology) are not performing the water quality functions expected of natural wetlands. Results suggest that the legacy of prior land use may influence the activity and composition of wetland microbial communities. Our results have also generated hypotheses about the ecological drivers that are most important for shaping microbial community structure and function in floodplain forest wetlands. Soil moisture, soil organic matter concentration, and available nitrogen were highly correlated to microbial community structure and denitrification function. Establishing the link between these ecological drivers and the functions carried out by wetland microbial communities will help to develop and refine assessment and monitoring tools that are closely related to the delivery of ecosystem services. We have demonstrated that denitrification rates are significantly lower in constructed/restored wetlands. Though some of the constructed wetland sites meet the criteria for jurisdictional wetlands, they are not currently performing some of the water quality functions expected of natural wetlands. This suggests that criteria for wetland classification may not be relevant to determining the successful restoration of denitrification and other microbially-mediated processes. These findings have important implications for water quality as well. Our findings suggest that wetlands constructed under current wetland assessment criteria will not likely replace the nutrient removal function of natural wetlands. Dissemination of our results (through publications, conference proceedings, and news articles) should lead to recommendations to improve wetland assessment.

Publications

  • Matthews, J.W., Peralta, A.L., Soni, A., Baldwin, P., Kent, A.D. and Endress, A.G. 2009. Local and landscape correlates of non-native species invasion in restored wetlands. Ecography 32:1031-1039.
  • Matthews, J.W., Peralta, A.L., Flanagan, D.N., Baldwin, P., Soni, A., Kent, A.D. and Endress, A.G. 2009. Relative influence of landscape vs local factors on plant community assembly in restored wetlands. Ecological Applications 19:2108-2123.


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

Outputs
OUTPUTS: New wetland regulations recently passed by the U.S. Army Corps of Engineers require developers to meet stricter guidelines in mitigation planning, implementation, and management to increase restoration success. It was specifically noted that functional assessment methods must be refined, and the scientific community was tasked with development of reliable, ecologically-based performance standards for wetlands. Despite decades of wetland restoration, there is continued uncertainty in restoration trajectories, and more information is necessary to provide better monitoring and assessment of wetland development. This research will help to improve wetland assessment protocols to include below-ground processes that affect water quality. We are using the data collected in this research to generate models of soil chemical and physical variables and plant community variables that influence denitrification data. Outputs of this research will include recommendations for soil parameters that could be included in assessment and monitoring approaches. Results from these wetland microbial ecology surveys were presented to wetland professionals and researchers at several national and international meetings: Flanagan, D.N., Matthews, J.W. and Kent, A.D. Can constructed wetlands replace natural wetlands A study into microbial community structure and function in constructed vs. natural wetlands. Poster presentation at the Illinois State Academy of Sciences conference, Urbana, Illinois. April 4-5, 2008. Poster presentation at the Society of Wetland Scientists 2008 Conference, Washington, DC. May 27-30, 2008. Peralta, A.L., Matthews, J.W. and Kent, A.D. Wetland mitigation bank assessment from a soil microbial ecology perspective. Poster presentation at the 100th Annual Meeting of the Illinois State Academy of Sciences, Urbana, Illinois. April 4-5, 2008. Kent, A.D., Peralta, A.L., Flanagan, D.N. and Matthews, J.W. Wetland assessment from a soil microbial ecology perspective. Oral presentation at the 2008 ASLO Summer Meeting, St. John's, Newfoundland, Canada. June 8-13, 2008. Peralta, A.L., Flanagan, D.N., Matthews, J.W., Endress, A.G. and Kent, A.D. A microbial community ecology approach to wetland mitigation assessment. Poster presentation at the 93rd Ecological Society of America Annual Meeting, Milwaukee, Wisconsin. August 3-8, 2008. Poster presentation at the 12th International Symposium on Microbial Ecology, Cairns, Australia. August 17-22, 2008. A M.S. thesis was also produced from this work: D.N. Flanagan. 2009. Microbial community structure and function in wetland ecosystems. Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign. Results were also disseminated through the University of Illinois news service: http://www.nres.uiuc.edu/Dynamic.aspxPageId=124&ContentObjectId=1303 &ContentObjectName=Microbes+in+Natural+Illinois+Wetlands+Protect+Wate r+Quality. Teaching materials were developed from this wetland microbial ecology work and incorporated into a Soil Microbiology course taught by the PI. PARTICIPANTS: Angela Kent: PI, project management, experimental design. Diana Flanagan: MS student, conducted fieldwork in 2006 and 2007, received training in denitrification assays, soil chemistry analyses, DNA extraction, molecular microbial community analyses. Diana presented the results of her research at state and national conferences. Ariane Peralta: technician/PhD student, conducted field work at Grundy County site in 2007, received training in soil chemistry analyses, denitrification assays, nitrification assays, DNA extraction, molecular microbial community analyses. Additional field work was conducted in 2008 at The Nature Conservancy's Emiquon wetland restoration site near Havana, Illinois to monitor wetland microbial activity and composition in relation to fluctuating water levels during the initial stages of wetland restoration. Ariane presented the results of her research at state, national, and international conferences. Ariane received the "best poster" award at the 2008 Illinois State Academy of Science meeting, and also at the 2008 International Society of Microbial Ecology meeting in Cairns, Australia. Jeff Matthews: collaborator with the Illinois Natural History Survey, provided access to wetland sites and plant community data. Jeff presented the results of his research at state, and national conferences. He received the "best oral presentation" award at the 2008 Illinois State Academy of Science meeting. Partnerships were developed with wetland researchers at the University of Illinois at Springfield, the Emiquon Field Station, and The Nature Conservancy. TARGET AUDIENCES: Wetland researchers, natural resource managers, extension professionals, undergraduate students interested in conservation of natural resources, landowners, as well as public, state and federal agencies concerned with natural resources. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our results from wetland samples collected from throughout Illinois demonstrate that composition and denitrification activity of microbial communities in constructed wetlands differs significantly from the microbial communities found in natural wetlands. Denitrification rates are significantly lower in constructed/restored wetlands. Though some of the constructed wetland sites meet the criteria for jurisdictional wetlands, they are not currently performing some of the water quality functions expected of natural wetlands. This indicates that current criteria for wetland classification may not be relevant to determining the successful restoration of denitrification and other microbially-mediated processes. This has important implications for water quality as well. Wetlands constructed under current wetland assessment criteria will not likely replace the nutrient removal function of natural wetlands. Understanding the ecology of specific microbial populations involved in important wetland functions like denitrification will lead to a better understanding of the factors that influence such processes, and will aid in the design of wetland construction and management practices that will optimize microbial denitrification, reducing eutrophication and degradation of coastal ecosystems. We will also improve our capacity to predict the effect of land use changes on microbially-mediated processes. Our results indicate that currently used wetland assessments do not adequately predict restoration of microbial nutrient removal functions. Dissemination of our results (through publications, conference proceedings, and news articles) should lead to recommendations to improve wetland assessment.

Publications

  • Matthews, J.W. and Endress, A.G. 2008. Performance criteria, compliance success, and vegetation development in compensatory mitigation wetlands. Environmental Management 41:130-141.
  • Matthews, J.W., Peralta, A.L., Flanagan, D.N., Baldwin, P., Soni, A., Kent, A.D. and Endress, A.G. 2009. Relative influence of landscape versus local factors on plant community assembly in restored wetlands. (Under Review).
  • Peralta, A.L., Matthews, J.W. and Kent, A.D. 2009. Improving functional assessment of wetland restoration from a microbial community approach. (Submitted).


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

Outputs
OUTPUTS: Denitrification is among the most important of the ecosystem services provided by microbial communities in wetland sediments. Loss of this function is a serious consequence of land use change in Illinois. Little is known about the relationship between microbial community structure and ecosystem processes, or about the influence of environmental factors and land use change on bacterial community composition. This research examines the link between microbial community composition and denitrification in natural, restored and constructed wetlands, in order to improve our understanding of the ecological factors that control denitrification. A survey of 31 natural and constructed wetlands was carried out in the summer of 2006 in collaboration with the Illinois Natural History Survey. Denitrification assays were carried out for each sample, and data on vegetation, hydrology, soil moisture, pH, total and available N, and organic carbon were collected. During 2007, two similar datasets were generated. One study examined 6 sets of paired natural and constructed wetlands representing 3 different hydrological settings typical of Illinois wetlands. A second 2007 study was carried out in the 342 ha Morris Wetland Mitigation Bank in Morris, IL (Grundy County). This mitigation bank has five areas containing wetland vegetation and hydric soils. Within each wetland area, variable hydrology has resulted in local sites that were considered "successful" and "failed" examples of wetland restoration. Soil samples were collected from "successful" and "failed" sites in each of the wetland areas and from paired "reference" sites located in natural floodplain forests adjacent to each constructed wetland. In each study, DNA was extracted from each soil sample for characterization of microbial community composition using both functional and phylogenetic assessments. 16S rRNA T-RFLP community fingerprints are allowing us to compare microbial community structure across wetlands that differ in efficiency of nutrient removal. Diversity and relative abundance of dentrifiers among sites will be evaluated using T-RFLP on a functional gene (nosZ) involved in dentrification. Populations or assemblages that are associated with effective nitrate removal will be identified, and multivariate analyses will be used to determine biotic and abiotic factors that influence the distribution of these populations. Clone libraries containing 16S rRNA genes and phylogenetic predictions from T-RFLP analysis programs will be used identify specific populations. The results of these three wetland surveys will generate hypotheses about microbial populations associated with wetland denitrification that will be tested in future fieldwork, and in mesocosm experiments where the response of microbial communities will be monitored as environmental factors of interest are manipulated. Teaching materials were developed from this wetland microbial ecology work and incorporated into a Soil Microbiology course taught by the PI. Results from these wetland microbial ecology surveys were presented at the International Symposium on Soil Biodiversity and Ecology in Taipei, Taiwan, September 9-13, 2007. PARTICIPANTS: Angela Kent: PI, project management, experimental design. Diana Flanagan: MS student, conducted fieldwork in 2006 and 2007, denitrification assays, soil chemistry analyses, DNA extraction, molecular microbial community analyses. Ariane Peralta: technician/PhD student, conducted field work at Grundy County site in 2007, soil chemistry analyses, denitrification assays, DNA extraction, molecular microbial community analyses. Jeff Matthews: collaborator with the Illinois Natural History Survey, provided access to wetland sites and plant community data.

Impacts
Microbial communities in riparian wetlands carry out biogeochemical transformations that are important for improving water quality by decreasing the concentration of nutrients in runoff. The Clean Water Act requires wetland restoration or creation to mitigate wetland loss due to land use change. The policy of wetland mitigation assumes that restoration outcomes follow a predictable trajectory, but we have limited ability to predict restoration site development or restoration of water quality functions, in part because few studies examine the ecology of microbial communities in natural or constructed wetlands. Results from the study conducted at the Grundy County mitigation bank demonstrate that composition and denitrification activity of microbial communities in constructed wetlands differs significantly from the microbial communities found in natural wetlands. Though some of the constructed wetland sites meet the criteria for jurisdictional wetlands, they not currently performing some of the water quality functions expected of natural wetlands. This indicates that current criteria for wetland classification may not be relevant to determining the successful restoration of denitrification and other microbially-mediated processes. Understanding the ecology of specific microbial populations involved in important wetland functions like denitrification will lead to a better understanding of the factors that influence such processes, and will aid in the design of wetland construction and management practices that will optimize microbial denitrification, reducing eutrophication and degradation of coastal ecosystems. We will also improve our capacity to predict the effect of land use changes on microbially-mediated processes.

Publications

  • Peralta, A.L., Matthews, J.W., Flanagan, D.N. and Kent, A.D. 2007. Microbial community structure and function in restored floodplain forest wetlands. Proceedings of the International Symposium on Soil Biodiversity and Ecology. Taipei, Taiwan.


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

Outputs
Denitrification is among the most important of the ecosystem services provided by microbial communities in wetland sediments. Loss of this function is a serious consequence of land use change in Illinois. Microbial communities and their activities in wetland ecosystems are regularly treated as a simplistic "black box" where only the inputs and outputs are examined. Little is known about the relationship between microbial community structure and ecosystem processes, or about the influence of environmental factors and land use change on bacterial community composition. This research examines the link between microbial community composition and denitrification in natural, restored and constructed wetlands, in order to improve our understanding of the ecological factors that control denitrification. A survey of 36 natural and constructed wetlands was carried out in the summer of 2006 in collaboration with the Illinois Natural History Survey. Respiration and denitrification assays were carried out for each sample, and data on vegetation, hydrology, soil moisture, pH, total and available N, and organic carbon were collected. We are in the process of completing the activity assays, and soil chemical and physical analyses. We will soon begin extracting DNA from archived samples collected at each wetland. Microbial community composition in the archived samples will be evaluated using both functional and phylogenetic assessments. ARISA community fingerprints will allow comparisons of microbial community structure and diversity across wetlands that differ in efficiency of nutrient removal. Diversity and relative abundance of dentrifiers among sites will be evaluated using T-RFLP and quantitative PCR on functional genes (nos, nir) involved in dentrification. Populations or assemblages that are associated with effective nitrate removal will be identified, and multivariate analyses will be used to determine biotic and abiotic factors that influence the distribution of these populations. Clone libraries combining 16S rRNA genes and the 16S-23S rRNA intergenic spacer region (the region examined in ARISA fingerprints) will be used to identify specific populations. The results of this statewide survey will generate hypotheses about microbial populations associated with wetland denitrification that will be tested in future fieldwork, and in mesocosm experiments where the response of microbial communities will be monitored as environmental factors of interest are manipulated.

Impacts
Microbial communities in riparian wetlands carry out biogeochemical transformations that are important for improving water quality by decreasing the concentration of nutrients in runoff. The Clean Water Act requires restoration or creation to mitigate wetland loss due to anthropogenic activity. The policy of wetland mitigation assumes that restoration outcomes follow a predictable trajectory, but we have limited ability to predict restoration site development, in part because few studies examine the ecology of microbial communities in natural or constructed wetlands. Understanding the ecology of specific microbial populations involved in important wetland functions like denitrification will lead to a better understanding of the factors that influence such processes, and will aid in the design of wetland management practices that will reduce eutrophication and degradation of coastal ecosystems. We will also improve our capacity to predict the effect of land use changes on microbially-mediated processes.

Publications

  • No publications reported this period