Progress 08/01/03 to 07/31/07
Outputs
OUTPUTS: The project was carried out by the PI's (F. Garcia-Pichel and S. Neuer), two undergraduates, one graduate student and one technician. The junior members of this team received significant teaching and mentoring, in some cases carreer-determining, as direct outputs of this project. We also sought collaborations with groups in Los Alamos National Lab to complement research on unexpected findings, and in Cornell Universtity to work onmethods developments.These have developed into truly colaborative relationships. The bulk of our activities, however, were geared towards conducting and analyzing experiments designed to test our originally conceived dual goal to falsify or prove the hypothesis that the nitrogen cycling capacity of soil crusts increases with maturity, and to demonstrate that soil crusts export nitrogenous coumpounds to subsurface soils. The outcomes of this investigation were disseminated through standard scientific publications (see below), and through direct contributions to targeted audiences, including presentations by the PI's and students in several avenues. Students presented contributions to American Society for Microbiology meetings in 2003, 2005, to the National Astrobiology Meeting in 2003, to the International Microbial Ecology (ISME) meeting in 2004. The PI gave invited talks in the ISME meeting in 2006, as well as in the PI meeting for the Soils Program, of CRIS in and 2005, and 2007, and in the yearly meeting of the Spanish society for Microbiology in 2003. An opening lecture on the reulst of this was given by the PI at the 2006 International Symposium of Photosynthetic Porkaryotes, and invited seminars were given at the University of Arizona, and at University of California-Merced. A round table discussion of desert microbiology was organized by the PI at the International Society for Microbial Ecolog, 10th Congress. Cancun, Mexico. In terms of direct dissemination to the public, results and themes of this proposal have been integrated into a public-oriented web-page (http://www.public.asu.edu/~ferran/C%20and%20N.html, and a podcast on soils and soilcrusts directed to younger audiences has been prepared and is available online, within the frame of ASU's Ask-a-Biologist:(http://askabiologist.asu.edu/podcasts/mp3_files/ask_ a_biologist_vol_022.mp3) PARTICIPANTS: PIs: Dr. Ferran Garcia-Pichel, Dr Susanne Neuer Doctoral Student: Shannon L. Johnson Technicians: Ruth Potrafka and Ian Anderson Undergraduates: Ian Anderson, Charles Budinoff Collaborators: Los Alamos: Dr. Chris Yeager, Dr. Cheryl Kuske. USGS: Dr Jayne Belnap; Cornell U. : Dr. Dan Buckley TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We established the relative contribution of ammonium uptake, volatilization, and nitrification as competing sinks for the efflux of ammonium from soil crusts, using a combination of vertically explicit determinations of N fixation and potential nitrification at sub-cm resolution in crusts during activity pulse events. N fixation rates were high , but potential areal ammonium oxidation (AO) rates were commensurate with inputs, indicating that AO was the major sink for N in the crusts. AO (hence, nitrification) was a spatially restricted but important process in BSC nitrogen cycling. Proteobacterial diversity was rather restricted, with very clear dominance by members such as Nitrosospira. This was confirmed by obtaining cultivated isolates of Nitrosospira that shared this group of amoA genes. We also surveyed potential archaeal ammonia oxidizers, finding that crusts contain essentially one single phylotype of "archaeal amoA" gene. The archaeal diversity measured in terms of 16S rRNA phylotypes is larger than that of archaeal amoA, with 2-3 distinct phylotypes being common. Quantitative PCR showed that populations of archaeal and bacterial ammonia oxidizers may be of comparable magnitude. Our second stated goal was to assess the importance and extent of denitrification in soil crusts. This had been a controversial issue regarding the biology of soil crusts Using a combination of microscale and mesoscale process-rate determinations, we found that, in spite of theoretically optimal conditions, denitrification rates in BSC were paradoxically irrelevant for nitrogen cycling. Denitrifier populations within BSCs were accordingly extremely low. Because of the absence of denitrification, and because of the limitation of respiration and ammonia-oxidation imposed by diffusive O2 supply, we could demonstrate that BSCs export ammonium, nitrate and organic-N in approximately stoichiometrically equal proportions to subsurface soils. Furthermore, the net export rate was commensurate with that of N2-fixation. We found that N2-fixation rates in mature crusts peaked close to the crust surface where populations of heterocystous cyanobacteria dominate. This was expected. However, in developmentally early crusts, nitrogen fixation was measurable and important deep within the crusts, where light does not penetrate. This clearly precluded phototrophs as virtually exclusive contributors to nitrogen fixation during the early stages of crust formation. N fixation must have been consistently underestimated in the past, because of a procedural shortcoming that did not allow sufficient diffusion time to detect deep-sitting activity. We needed to address this possibly significant lack of knowledge. In order to find out if we could identify the bacteria responsible for this activity, we teamed up with colleagues at Los Alamos National Lab to study the community composition of potential fixers through clone library surveys of nifH genes . Indeed, while most sequences found are cyanobacterial in origin, approximately 10% correspond to heterotrophic bacteria. Implementing novel techniques such as stable isotopic labeling (SIP) will be necessary in the future.
Publications
- Garcia-Pichel, F., Johnson, S.L, Youngkin,D. and Belnap,J. (2003). Small-scale vertical distribution of bacterial biomass and diversity in biological soil crusts from arid lands in the Colorado Plateau. Microbial Ecology 46:312-321
- Garcia-Pichel, F, J. Belnap, S. Neuer, F Schanz (2003) Estimates of global cyanobacterial biomass and its distribution. Archive for Hydrobiology/Algological Studies 109: 213-228
- Johnson, S.L., Budinoff, C.R., Belnap, J., Garcia-Pichel, F (2005). Relevance of ammonium oxidation in biological soil crust communities. Environmental Microbiology 7:1-12
- Pringault, O, Garcia-Pichel, F. (2004). Hydrotaxis of cyanobacteria in desert crusts. Microbial Ecology 47:363-373
- Johnson, S L, Neuer, S, & Garcia-Pichel F (2007) Export of nitrogenous compounds due to incomplete cycling within biological soil crusts. Environm. Microbiol. 9: 680-689
- Yeager, C M, Kornosky, J L, Morgan, R L,. Cain, E C, Belnap. J, Garcia-Pichel, F and Kuske C R (2007). Three distinct clades of cultured heterocystous cyanobacteria constitute the dominant N-fixing members of biological soil crusts of the Colorado Plateau, USA. FEMS Microbiol Ecol 60 (1): 85-97
- Chorover, J., R Kretschmar, F Garcia-Pichel, D Sparks (2007) Soil Biogeochemical Processes within the critical zone. Elements 3: 321-326.
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Progress 10/01/05 to 09/30/06
Outputs
Our work on ammonia oxidizers was ready to be submitted (see previous report) when the unexpected discovery of archaeal ammonia oxidizers was made public. This did not invalidate our data, but it raised the possibility that we may have been missing a significant part of the nitrifying community. We decided to complement the work with a survey of potential archaeal ammonia oxidizers, currently being finished. We have found that all crusts contain essentially one single phylotype of "archaeal amoA" gene. The archaeal diversity measured in terms of 16S rRNA phylotypes is larger than that of archaeal amoA, with 2-3 distinct phylotypes being common. Quantitative PCR measurements point out that the populations of both archaeal and bacterial ammonia may be of comparable magnitude. In the absence of relevant cultivated archaeal isolates , alternative methodologies to relate structure and function, such as stable isotopic labeling, may be necessary to attribute the relative
contributions of bacteria and archaea to the cycling of reduced nitrogen. We found that N2-fixation rates in mature crusts expectedly peaked close to the crust surface where populations of heterocystous cyanobacteria dominate. However, in developmentally early crusts, nitrogen fixation was measurable and important deep within the crusts, where light does not penetrate. This clearly precluded phototrophs as virtually exclusive contributors to nitrogen fixation and concluded that nitrogen fixation must have been consistently underestimated in the past. In order to find out if we could identify the bacteria responsible for this activity, we studied the community composition of potential fixers through clone library surveys of nifH genes. Indeed, while most sequences found are cyanobacterial in origin, approximately 10% correspond to heterotrophic bacteria, particularly those that can be assigned to ?-proteobacterial-type nifH. In the absence of appropriate cultivated isolates, it is at
this point impossible to determine more specifically the identity of non-photosynthetic fixers using these techniques, without implementing novel techniques such as stable isotopic labeling (SIP). Stable isotope probing (SIP) of nucleic acids offers a means to study the metabolic activity of microorganisms as they occur in the environment and to characterize novel organisms that may have escaped detection previousl. In fact, we initiated SIP labeling experimens that clearly demonstrate its value. After a 3 day incubation under 15N2, DNA extracts from light crusts display an increase in density in CsCl gradients with respect to BSC incubated under 14N2. DGGE finger-prints of the heavy DNA fractions (shaded area in left panel), shown in the gel images on the right, demonstrate that some microbes (bands with arrow) are present only in the 15N treatment but not in the control. These microbes are thus identified as nitrogen fixers. Sequencing of the bands in question revealed their
identity: Frankia, Geodermatophilus and other allied actinobacteria.
Impacts
The main impact statement remains essentially unchanged, but strengthened
Publications
- Johnson, S L, Neuer, S, & Garcia-Pichel F (2007) Export of nitrogenous compounds due to incomplete cycling within biological soil crusts. Environm. Microbiol. 9: 680-689
- Yeager, C M , Kornosky, J L, Morgan, R L,. Cain, E C, Belnap. J, Garcia-Pichel, F and Kuske C R (2007). Three distinct clades of cultured heterocystous cyanobacteria constitute the dominant N2-fixing members of biological soil crusts of the Colorado Plateau, USA. FEMS Microbiology Ecology, Online Early Articles: 24-Jan-2007 doi: 10.1111/j.1574-6941.2006.00265.
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Progress 10/01/04 to 09/30/05
Outputs
On the one hand, we pursued the question of lack of denitrification further, given its key importance for nitrogen cycling. We could successfully validate results obtained the previous year in all respects with a second set of samples. In addition, we carried out measurements of trace metal availability (Molybdenum and Vanadium), to discount a trace metal limitation on enzyme activity. It was also important to scale up these micro-scale results to the level at which most soil scientists and rangeland managers work Therefore, we tested the prediction, based on microscale results, that there should be a net export of approximately stoichiometric ammonium nitrate with each precipitation pulse in soil crusts. This we did in a set of rain-simulation incubations using 10 cm sample cores. The results confimed our hypothesis fully. A manuscript including all of these results is under review presently. On the other hand we pursued further the investigation of the nature and
distribution of ammonia- oxidizing bacteria in the crusts. Here we found that a novel group of previously unrecognized Nitrosospira-like bacteria are responsible for the bulk of nitrogen oxidation in soil crust. These are distributed widely through crusts in the western US, even though clear biogeographical separation is seen between Northern (cold) and Southern (hot) deserts. A manuscript presenting these results is being finalized and will be submitted shortly. Finally we started our experimental approach to use a combination of molecular ecology with N15 tracers. Here, direct isotope measurements are used to determine rates of N-transformations, and SIP (stable isotope probing) is used to reveal the identity of the major organisms involved in those transformation. Preliminary results on simple incubations have yielded encouraging results, but the technique will still need to be optimized in further trials.
Impacts
The main impact statement remains essentially unchanged, but strangthened / confirmed with a second data set and validated at larger spatial scales.
Publications
- S.L. Johnson (2005). Microscale nutrient cycling in biological soil crusts of the Colorado Plateau. Ph.D Thesis. Arizona State University, Tempe AZ, 366 pp.
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Progress 10/01/03 to 09/30/04
Outputs
We proceeded to study the importance of denitrification for internal N cycling within the crust bounds. Efforts were geared first to test and optimize the construction of nitrate microsensors to measure profiles of nitrate concentration within the crust during simulated rain events. This was successfully accomplished for various crust samples (from simple to complex biologically speaking) . The patterns of nitrate obtained are consistent with the important role of ammonia oxidizers, and are also congruent with profiles of ammonium, oxygen and pH obtained in parallel. The totality of microchemical profiles obtained give a very detailed picture of the geochemical environment in the soil solution of soil crusts in which to interpret biological activity measurements. There was not much evidence for localized nitrate consumption, but rather just for production and diffusive export. Methodology for biogeochemical rate measurements for denitrification were adapted from the
literature to suit small scale incubations, and were used to gauge the strength of denitrification within soil crusts in a variety of samples, seasons and incubation conditions. In all cases denitrification was measurable, but surprisingly not really comparable to rates of nitrogen fixation or ammonia oxidation by orders of magnitude. Population analyses using both molecular and cultivation efforts indicate that indeed nitrate reduction to the level of N2 is not a significant process in crust, with denitrifyer populations much lower than in many soils. Reasons for this unexpected break in the N-cycle are being currently investigated. These results are being prepared for publication.
Impacts
The single most important impact of the results the apparent the lack of denitrification This single finding implies that the crusts are acting as net exporters of excess ammonium and nitrate as dissolve chemical species to deeper soils through flushing by rain events, rather than back to the atmosphere, and are thus an important factor in the fertility of arid land soils
Publications
- Johnson, S.L., Budinoff, C.R., Belnap, J., Garcia-Pichel, F (2005). Relevance of ammonium oxidation in biological soil crust communities. Environmental Microbiology 7:1-12
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