Bedard-Haughn, A.

Farrell, R.E.

Knight, J.D.

Lemke, R.

Sather, M.

Siciliano, S.

English

University of Saskatchewan

Does hydrogen evolution from HUP- field pea nodules stimulate nitrous oxide production?

eCommons@USASK

University of Saskatchewan Research Archive

Does Hydrogen Evolution From HUP- Field Pea Nodules Stimulate Nitrous Oxide Production? M. Sather1, R. Farrell1, J.D. Knight1, R. Lemke2, A. Bedard-Haughn1, S. Siciliano1  1 Department of Soil Science, University of Saskatchewan, Saskatoon, SK, S7N 5A8 2 Agriculture and Agricultural Food Canada (AAFC), Saskatoon, SK, S7N 0X2  Key Words: field pea, biological nitrogen fixation, Rhizobium, hydrogenase uptake  Introduction  Legumes, such as Pisum sativum (field pea), can fix atmospheric N2 into plant available forms through symbiosis with Rhizobium bacteria.  During biological nitrogen fixation (BNF), hydrogen gas (H2) is produced as a byproduct (Dong and Layzell, 2001). If the legume-Rhizobium symbiosis possesses the hydrogenase uptake enzyme (HUP+), the H2 gas produced can be recovered.  However, if the HUP enzyme is lacking (HUP-), the H2 gas produced through BNF diffuses into the rhizosphere (Dong and Layzell, 2001).  Hydrogen gas may cause conditions in the rhizosphere to change in a way that favours nitrous oxide (N2O) production.  These changes could include enhancing O2 consumption and increasing CO2 fixation; both of these conditions may favour denitrification and increase the potential for N2O production (Golding and Dong, 2010).   Nitrous oxide is a greenhouse gas that has a negative impact on the atmosphere by increasing radiative forcing and catalyzing ozone destruction.  The majority of N2O emissions in Canada are from agricultural sources; however, it is still unknown where much of the N2O is produced in the soil-plant system (Golding and Dong, 2010).  The objectives of this preliminary study were to analyze Rhizobium leguminosarum strains to identify HUP+ and HUP- strains using field pea nodules.  And secondly, to analyze H2 and N2O concentrations from pea roots and nodules grown with different R. leguminosarum strains as inoculants to determine if HUP- symbiosis, in the absence of soil, produces greater concentrations of H2 and N2O.  Materials and Methods   The first experiment used the methylene blue reduction assay to determine the HUP status of field pea nodules inoculated with various strains of R. leguminosarum (Lambert et al., 1985).  The four Rhizobium strains selected for this experiment were 128C52, 128C79, 128C53, and PJB5J1.   Strains 128C53 and PJB5J1 are largely isogenic except for the HUP enzyme.  Pea plants were grown in sterile sand for four weeks with N-free nutrient media.  The assay was conducted on nodules from the pea plants.  The second experiment conducted was focused on the H2 and N2O concentrations produced from pea roots.  Pea seeds were inoculated with one of six inoculation treatments.  The treatments included the four Rhizobium strains from the first experiment (128C52, 128C79, 128C53 and PJB5J1) and two control treatments, B151 (non-nodulating Rhizobium strain, largely isogenic with 128C53 and PJB5J1) and sterile water. Pea plants were grown for four weeks in sterile sand with N-free nutrient media.  After four weeks, roots were washed and then sealed in glass media jars with lids fitted with septa for gas sampling.  Half of the replicates for each treatment were given an ambient air atmosphere, the other half of the replicates had 120 ppm H2 injected into the jar to simulate a pre-existing H2 atmosphere in the rhizosphere.  Gas samples were collected every 30 minutes for a total of 120 minutes.  The atmosphere was replaced in the jar after each sampling. Gas samples were analyzed for H2 and N2O concentrations.  Results  The methylene blue reduction assay measures the HUP status of legume nodules (Lambert et al. 1985). Nodules that are HUP+ are able to reduce the methylene blue solution, which results in white discolouration around the nodules (Figure 1).  Nodules that are HUP- are not able to reduce the methylene blue and undergo no visible colour change (Figure 1). Strains 128C52 and 128C53 underwent discolouration and are HUP+ R. leguminosarum strains.  Strains 128C79 and PJB5J1 are did not change colour and are HUP- R. leguminosarum strains.                                          Figure 1. Results of the methylene blue reduction assay.  Hydrogen concentrations from the six R. leguminosarum treatments under an ambient atmosphere were significantly different from one another (Figure 2).  However, the H2 results from the six treatments were not significantly different from one another under the elevated H2 (120 ppm) atmosphere (Figure 3).                                                      Figure 2. Hydrogen gas concentrations for roots and nodules under ambient atmosphere conditions (P< 0.05, n=4).                                                Figure 3. Hydrogen gas concentrations from six inoculation treatments under elevated H2 atmosphere (P< 0.05, n=4).  0102030gHgrootbiomass-1h-10102030gHgrootbiomass-1h-120 40 60 80 100 1200102030Elapsed time (min)gHgrootbiomass-1h-120 40 60 80 100 120Elapsed time (min)128C52-1 128C53-1128C79-1B151-1Control-1PJB5J1-1020406080gHgrootbiomass-1h-1020406080gHgrootbiomass-1h-120 40 60 80 100 120020406080Elapsed time (min)gHgrootbiomass-1h-120 40 60 80 100 120050100Elapsed time (min)gHgrootbiomass-1h-1128C52-2 128C53-2128C79-2B151-2Control-2PJB5J1-2Nitrous oxide emissions were also measured in conjunction with the H2 concentrations from the six inoculation treatments under both the ambient air atmosphere and the elevated H2 atmosphere. The N2O concentrations under ambient atmosphere were not distinguishable from ambient N2O concentrations (results not shown).  The N2O results under an elevated H2 atmosphere had no significant differences between treatments (Figure 4).                                                   Figure 4. Nitrous oxide concentrations from roots and nodules under an elevated H2 atmosphere (P< 0.05, n=4).  Discussion  The methylene reduction assay was successful at identifying HUP- and HUP+ R. leguminosarum strains.  These four strains along with the two control treatments, B151 and Control (sterile water) form the six inoculation treatments that were used in the second experiment and which will be used in future experiments as well.  Under ambient air conditions, H2 concentrations from128C79 and PJB5J1 were significantly different from one another and the other four treatments.  The results confirm that theses strains are HUP- because they are producing H2 and not recycling it.  The differences in treatment were not observed under the elevated H2 atmosphere, differences were possibly masked by the artificially elevated H2 atmosphere background levels.  There were no significant differences in the N2O concentrations measured under either atmosphere; this is an anticipated result because there is no direct biological pathway between H2 production and N2O production in legume BNF.     012345N 2OproductiongNgrootbiomass-1h-1012345N 2OproductiongNgrootbiomass-1h-120 40 60 80 100 120012345N 2OproductionElapsed time (min)gNgrootbiomass-1h-120 40 60 80 100 12002468N 2OproductionElapsed time (min)gNgrootbiomass-1h-1128C52 128C53128C79B151ControlPJB5J1Conclusion  In the absence of soil, H2 production from HUP- nodules does not stimulate N2O production.  The next step in this process is to conduct a similar experiment with soil present in the rhizosphere to see whether or not H2 creates conditions in the rhizosphere that favour denitrification and N2O production.  References  Dong, Z. and D.B. Layzell. 2001. H2 oxidation, O2 uptake and CO2 fixation in hydrogen treated soils. Plant and Soil 229: 1-12.  Golding, A. and Z. Dong. 2010. Hydrogen production by nitrogenase as a potential crop rotation benefit. Environ. Chem. Lett. 8: 101-121.  Lambert, G.R., F.J. Hanus, S.A. Russell and H.J. Evans. 1985. Determination of the Hydrogenase Status of Individual Legume Nodules by a Methylene Blue Reduction Assay. App. And Environ. Micro. 50: 537-539.   

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Does hydrogen evolution from HUP- field pea nodules stimulate nitrous oxide production?

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