DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation

Abstract

To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for use with soil/rhizosphere samples. We tested the impact of different carbon (C) sources, oxygen concentrations (O2), and incubation times on FLNF rates of two low-fertility Michigan soils via ARA and 15N2.FLNF rates were greatest with addition of a C cocktail, at low O2, and with 7-day incubations for both methods. FLNF via ARA was 1700x greater with a C cocktail versus glucose only and via 15N2 was 17x greater with a C cocktail compared to other C sources and no-C controls. Specific O2 optimum varied by method and site. A 7-day incubation was needed for the ARA, but a 3-day incubation was suitable for 15N2. Lastly, we confirm previously identified issues with the ARA of acetylene-independent ethylene production/consumption resulting in potential FLNF measurement error of 1.3–52.3 μg N g–1 day–1. Here, we present an optimized method for measuring potential FLNF in soil/rhizosphere samples which will allow for consistent and comparable FLNF rate measurements. Researchers should account for C source, O2, and incubation time when assessing FLNF and use the ARA method with caution.

Authors:
ORCiD logo [1];  [2];  [3];  [1]
  1. Michigan State Univ., East Lansing, MI (United States)
  2. Michigan State Univ., Hickory Corner, MI (United States)
  3. Washington State Univ., Pullman, WA (United States)
Publication Date:
Research Org.:
Michigan State Univ., East Lansing, MI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1637663
Grant/Contract Number:  
SC0018409; FC02-07ER64494; SC0014108
Resource Type:
Accepted Manuscript
Journal Name:
Plant and Soil
Additional Journal Information:
Journal Volume: 445; Journal Issue: 1-2; Journal ID: ISSN 0032-079X
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; free-living nitrogen-fixation; 15N2 incorporation; acetylene reduction; Rhizosphere

Citation Formats

Smercina, Darian N., Evans, Sarah E., Friesen, Maren L., and Tiemann, Lisa K. Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation. United States: N. p., 2019. Web. doi:10.1007/s11104-019-04307-3.
Smercina, Darian N., Evans, Sarah E., Friesen, Maren L., & Tiemann, Lisa K. Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation. United States. https://doi.org/10.1007/s11104-019-04307-3
Smercina, Darian N., Evans, Sarah E., Friesen, Maren L., and Tiemann, Lisa K. Fri . "Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation". United States. https://doi.org/10.1007/s11104-019-04307-3. https://www.osti.gov/servlets/purl/1637663.
@article{osti_1637663,
title = {Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation},
author = {Smercina, Darian N. and Evans, Sarah E. and Friesen, Maren L. and Tiemann, Lisa K.},
abstractNote = {To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for use with soil/rhizosphere samples. We tested the impact of different carbon (C) sources, oxygen concentrations (O2), and incubation times on FLNF rates of two low-fertility Michigan soils via ARA and 15N2.FLNF rates were greatest with addition of a C cocktail, at low O2, and with 7-day incubations for both methods. FLNF via ARA was 1700x greater with a C cocktail versus glucose only and via 15N2 was 17x greater with a C cocktail compared to other C sources and no-C controls. Specific O2 optimum varied by method and site. A 7-day incubation was needed for the ARA, but a 3-day incubation was suitable for 15N2. Lastly, we confirm previously identified issues with the ARA of acetylene-independent ethylene production/consumption resulting in potential FLNF measurement error of 1.3–52.3 μg N g–1 day–1. Here, we present an optimized method for measuring potential FLNF in soil/rhizosphere samples which will allow for consistent and comparable FLNF rate measurements. Researchers should account for C source, O2, and incubation time when assessing FLNF and use the ARA method with caution.},
doi = {10.1007/s11104-019-04307-3},
journal = {Plant and Soil},
number = 1-2,
volume = 445,
place = {United States},
year = {2019},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 14 works
Citation information provided by
Web of Science

Figures / Tables:

Table 1 Table 1: Site characteristics including soil properties, climate, and land use history.

Save / Share:

Works referenced in this record:

Global nitrogen budgets in cereals: A 50-year assessment for maize, rice and wheat production systems
journal, January 2016

  • Ladha, J. K.; Tirol-Padre, A.; Reddy, C. K.
  • Scientific Reports, Vol. 6, Issue 1
  • DOI: 10.1038/srep19355

Soils of the GLBRC Marginal Land Expeirment (MLE) Sites
text, January 2018


Effects of oxygen, mannitol and ammonium concentrations on nitrogenase [C2H2] activity in a marine skeletal carbonate sand
journal, January 1975


Rapid N^2 Fixation in Pines, Alder, and Locust: Evidence From the Sandbox Ecosystems Study
journal, March 1993

  • Bormann, Bernard T.; Bormann, F. Herbert; Bowden, William B.
  • Ecology, Vol. 74, Issue 2
  • DOI: 10.2307/1939318

Ethylene Biosynthesis and Signaling Networks
journal, May 2002

  • Wang, Kevin L. -C.; Li, Hai; Ecker, Joseph R.
  • The Plant Cell, Vol. 14, Issue suppl 1
  • DOI: 10.1105/tpc.001768

Impact of artificial root exudates on the bacterial community structure in bulk soil and maize rhizosphere
journal, September 2003


The Contamination of Commercial 15N2 Gas Stocks with 15N–Labeled Nitrate and Ammonium and Consequences for Nitrogen Fixation Measurements
journal, October 2014


Microbially Mediated Plant Functional Traits
journal, December 2011


Effect of Water-Filled Pore Space on Carbon Dioxide and Nitrous Oxide Production in Tilled and Nontilled Soils1
journal, January 1984


Evolution of Molybdenum Nitrogenase during the Transition from Anaerobic to Aerobic Metabolism
journal, March 2015

  • Boyd, Eric S.; Costas, Amaya M. Garcia; Hamilton, Trinity L.
  • Journal of Bacteriology, Vol. 197, Issue 9
  • DOI: 10.1128/JB.02611-14

The effect of organic amendment, water content, and oxygen on the incorporation of 15 N 2 by some agricultural and forest soils
journal, August 1969

  • Brouzes, R.; Lasik, J.; Knowles, R.
  • Canadian Journal of Microbiology, Vol. 15, Issue 8
  • DOI: 10.1139/m69-160

Functional Ecology of Free-Living Nitrogen Fixation: A Contemporary Perspective
journal, December 2011


Associative nitrogen fixation (ANF) in switchgrass (Panicum virgatum) across a nitrogen input gradient
journal, June 2018


Epoxidation of Ethylene by Whole Cell Suspension of Methylosinus trichosporium IMV 3011
journal, January 2017


Kinetics of nitrogen fixation in a glucose-amended, anaerobically incubated soil
journal, March 1973


Oxidation of ethylene by soil bacteria
journal, March 1976


Effect of oxygen partial pressure on nitrogen fixation and acetylene reduction in a sandy loam soil amended with glucose
journal, December 1971

  • Brouzes, R.; Mayfield, C. I.; Knowles, R.
  • Plant and Soil, Vol. 35, Issue 1
  • DOI: 10.1007/BF02661873

Differential Growth Responses of Soil Bacterial Taxa to Carbon Substrates of Varying Chemical Recalcitrance
journal, January 2011

  • Goldfarb, Katherine C.; Karaoz, Ulas; Hanson, China A.
  • Frontiers in Microbiology, Vol. 2
  • DOI: 10.3389/fmicb.2011.00094

To Fix or Not To Fix: Controls on Free-Living Nitrogen Fixation in the Rhizosphere
journal, January 2019

  • Smercina, Darian N.; Evans, Sarah E.; Friesen, Maren L.
  • Applied and Environmental Microbiology, Vol. 85, Issue 6
  • DOI: 10.1128/AEM.02546-18

Anaerobic processes in soil
journal, February 1984

  • Tiedje, J. M.; Sexstone, A. J.; Parkin, T. B.
  • Plant and Soil, Vol. 76, Issue 1-3
  • DOI: 10.1007/BF02205580

Use and abuse of the acetylene reduction assay for measurement of “associative” nitrogen fixation
journal, January 1987


The Significance of Nonsymbiotic Nitrogen Fixation
journal, March 1965


Nitrogen fixation and acetylene reduction in decaying conifer boles: effects of incubation time, aeration, and moisture content
journal, September 1982

  • Silvester, Warwick B.; Sollins, Phillip; Verhoeven, Thomas
  • Canadian Journal of Forest Research, Vol. 12, Issue 3
  • DOI: 10.1139/x82-098

The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists
journal, July 2014

  • Baldani, José Ivo; Reis, Veronica Massena; Videira, Sandy Sampaio
  • Plant and Soil, Vol. 384, Issue 1-2
  • DOI: 10.1007/s11104-014-2186-6

Possible contribution of alternative nitrogenases to nitrogen fixation by asymbiotic N2-fixing bacteria in soils
journal, February 2014


Non-Symbiotic Nitrogen Fixation in some Quebec Soils
journal, February 1965

  • Chang, P-C.; Knowles, R.
  • Canadian Journal of Microbiology, Vol. 11, Issue 1
  • DOI: 10.1139/m65-005

Methods for quantification of nitrogen fixation associated with gramineae
journal, January 1987


Diversity of Nitrogen-Fixing Bacteria Associated with Switchgrass in the Native Tallgrass Prairie of Northern Oklahoma
journal, July 2014

  • Bahulikar, Rahul A.; Torres-Jerez, Ivone; Worley, Eric
  • Applied and Environmental Microbiology, Vol. 80, Issue 18
  • DOI: 10.1128/AEM.02091-14

Oxygen inhibition of acetylene reduction (nitrogen fixation) in soil: Effect of glucose and oxygen concentrations
journal, December 1973


Evaluation of nitrogen fixation by bacteria in association with roots of tropical grasses.
journal, January 1980


An imaging method for oxygen distribution, respiration and photosynthesis at a microscopic level of resolution
journal, September 2012


The Acetylene-Ethylene Assay for N 2 Fixation: Laboratory and Field Evaluation
journal, August 1968

  • Hardy, R. W. F.; Holsten, R. D.; Jackson, E. K.
  • Plant Physiology, Vol. 43, Issue 8
  • DOI: 10.1104/pp.43.8.1185

The dynamics of oxygen concentration, pH value, and organic acids in the rhizosphere of Juncus spp.
journal, June 2011


Soil pH correlates with the co-occurrence and assemblage process of diazotrophic communities in rhizosphere and bulk soils of wheat fields
journal, June 2018


Root effects on the spatial and temporal dynamics of oxygen in sand-based laboratory-scale constructed biofilters
journal, September 2013


Natural formation of ethylene in forest soils and methods to correct results given by the acetylene-reduction assay
journal, January 1983


Acetylene reduction assay can overestimate nitrogen-fixation in soil
journal, January 1979


Identifying the microbial taxa that consistently respond to soil warming across time and space
journal, November 2016

  • Oliverio, Angela M.; Bradford, Mark A.; Fierer, Noah
  • Global Change Biology, Vol. 23, Issue 5
  • DOI: 10.1111/gcb.13557

Ethylene production and decomposition in soils
journal, April 1998

  • Zechmeister-Boltenstern, S.; Smith, Keith A.
  • Biology and Fertility of Soils, Vol. 26, Issue 4
  • DOI: 10.1007/s003740050388

Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation
journal, January 2014

  • Gupta, V. V. S. R.; Kroker, S. J.; Hicks, M.
  • Crop and Pasture Science, Vol. 65, Issue 10
  • DOI: 10.1071/CP14109

Oxygen and Hydrogen in Biological Nitrogen Fixation
journal, October 1980


Oxygen dynamics in the rhizosphere of Cymodocea rotundata
journal, January 1998

  • Pedersen, O.; Borum, J.; Duarte, Cm
  • Marine Ecology Progress Series, Vol. 169
  • DOI: 10.3354/meps169283

Maize germplasm chronosequence shows crop breeding history impacts recruitment of the rhizosphere microbiome
journal, March 2021


Soils of the GLBRC Marginal Land Expeirment (MLE) Sites
text, January 2018