ISSN 0147-6874, Moscow University Soil Science Bulletin, 2016, Vol. 71, No. 3, pp. 135–138. © Allerton Press, Inc., 2016. Original Russian Text © E.A. Soshnikova, A.S. Cherobaeva, A.L. Stepanov, E.V. Lebedeva, N.A. Manucharova, P.A. Kozhevin, 2016, published in Vestnik Moskovskogo Universiteta, Seriya 17: Pochvovedenie, 2016, No. 3, pp. 54–58.
New Processes of Microbial Transformation of Nitrogen in Soils as a Source of Greenhouse Gases E. A. Soshnikovaa*, A. S. Cherobaevab, A. L. Stepanova, E. V. Lebedevac, N. A. Manucharovaa, and P. A. Kozhevina a
Department of Soil Science, Moscow State University, Moscow, 119991 Russia b Center for Family Planning and Reproduction, Moscow, 117209 Russia c Vinogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, 119071 Russia *e-mail:
[email protected] Received November 15, 2015
Abstract—DNA isolation from soil samples and amplification of fragment of a key gene of nitrification, archaeal and bacterial amoA, revealed presence of the product in all investigated soil samples. Сharacteristics of ammonia-oxidizing microbial communities in agrocenoses and undisturbed soil were determined. Bacteria were predominant in agrocenoses (at circum-neurtal pH), whereas the share of representatives of domain Archaea (phylum Thaumarchaeota) increased in prokaryotic ammonia-oxidizing complexes of undisturbed forest ecosystems (at low pH). It was demonstrated that the contribution of taumarhaea in nitrous oxide emission from gray forest soil may reach 20–25%. Keywords: ammonia-oxidizing archaea, ammonia-oxidizing bacteria, soil, disturbance, nitrogen cycle DOI: 10.3103/S0147687416030078
INTRODUCTION Nitrification is one of the main sources of nitrous oxide in Earth’s modern atmosphere. The ecological role of this compound is that it has undergone photochemical transformations and forms intermediates that are involved in destruction of stratospheric ozone. Furthermore, nitrous oxide possessing a high capacity for absorption of reflected infrared radiation is one of the main greenhouse gases and has sufficient impact in global climate change. Due to the recent discovery of nitrifying archaea (Taumarchaeota) [3, 8, 14, 16, 17], their distribution and impact in nitrification in terrestrial and marine ecosystems are actively studied [5, 13, 20, 21]. It was revealed that archaea predominate over bacteria in various types of soil [6, 7, 9] but their role in formation and emission of nitrous oxide has not been studied. The goal of the present work was to study nitrifying microbial communities in sod-
podzolic and gray forest soils and estimate their impact in N2O formation. EXPERIMENTAL Samples of sod-podzolic and gray forest soils were objects of investigation. Their characteristics are shown in Table 1. DNA extraction was performed using Ultra Clean Soil DNA Isolation Kit (MoBio, Canada). Samples of the soils (0.5 g) were placed in 2 mL Bead Solution tubes with glass beads and vortexed. Then, 60 μL of S1 solution were added in the tubes. To perform polymerase chain reaction (PCR), 200 μL of IRS (Inhibitor Removing Solution) solution were added. The reaction mixture was vortexed for 10 min and centrifuged for 30 s at 10000 g. Supernatant was transferred in a pure microcentrifuge tube. Then, 200 μL of S2 solution were added and the mixture was vortexed
Table 1. General characteristics of soil samples Soil
Vegetation
Sod-podzolic moraine loam soil (forest) Mixed forest (spruce, birch) Sod-podzolic moraine loam soil (arable land) Barley Grey forest medium forest loam soil (forest) Deciduous forest (birch, aspen) 135
pHaqeous
Ctotal, %;
Nminimal, %
4.7 5.3 6.3
2.1 1.7 3.4
2.3 2.1 2.7
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for 5 s. Obtained solution was cooled at 4°C for 5 min. Supernatant was transferred in pure microcentrifuge tube without the sediment. Then, 1.3 mL of S3 reagent were added and the mixture was vortexed for 5 s. The obtained solution was transferred to a column filter and centrifuged for 1 min at 10000 g. Then, 300 μL of S4 reagent were added and centrifuged for 30 s at 10000 g. After passing the solution through the filter, centrifugation at 10000 g for 1 min was repeated. Colum filter was transferred into a pure 2 mL microcentrifuge tube, 50 μL of S5 reagent were placed in the center of white membrane filter, and filters were centrifuged at 10000 g for 30 s. The obtained sediment containing DNA was stored at –20°C for further analysis. DNA was purified using Wizard Genomic DNA Purification Kit (Promega, United States) according to the manufacturer’s recommendations. Volumes of all PCR reaction mixtures were 100 μL. PCR mixtures for 16S rRNA genes amplification contained 10 μL PCR buffer solution, 2 μL BSA, 2 μL dNTPs, 4 μL of 341F and 907R primers, 4 μL of DNA solution, 0.5 μL of Taq DNA polymerase, and 75.5 μL of H2O. PCR mixtures for amoA genes amplification contained 10 μL PCR buffer solution, 2 μL BSA, 4 μL dNTPs, 15 μL of amoAF and amoAR primers, 4 μL of DNA solution, 0.8 μL of Taq DNA polymerase, and 49.2 μL of H2O. Amplification was conducted using MyCycler thermal cycler (BioRad, United States). Gel electrophoresis was performed to detect products of PCR amplification. Samples of each PCR product (5 μL) were stained on a plate and transferred into a well for electrophoresis. Gel contained 75 mL of TE buffer solution (1x), 0.7 g of LE 2 agarose, and 1.3 μL of ethidium bromide. The following nutrient medium was used to isolate ammonia-oxidizing bacteria from sod-podzolic soil: 1.0 g/L (NH4)2SO4; 0.5 g/L K2HPO4; 2.0 g/L NaCl; 0.2 g/L MgSO4 · 7H2O; 0.05 g/L FeSO4 · 7H2O; 6.0 g/L CaCO3; and 1 mL trace elements solution. Two samples of soils collected from forest phytocenose and arable land were used for isolation of microorganisms. Each soil sample was inoculated in two flasks. One of them was incubated at ambient temperature, and the second one was incubated at 37°C. Cultures were grown for 1 month and passaged every 2 weeks. Growth of the cultures was tested every 7 days using Griess reagent. Production of nitrite (corresponding to AOB activity) was estimated by color intensity of mineral medium. After growth of microorganisms, samples of nutrient medium (1 mL of medium from each flask) were collected for DNA extraction and amplification. To estimate intensity of nitrous oxide emission, samples of the soils (5 g) were placed in penicillin flasks and moisturized with (NH4)2SO4 aqueous solution (0.3 mg of N/g of soil). The flasks were closed with rubber plugs and filled with acetylene (up to partial pressure of 10 Pa) to inhibit aerobic ammonia oxi-
dation by proteobacteria (i.e., activity of ammonia monooxygenase) [2]. This concentration of C2H2 does not inhibit the activity of thaumarchaea, because their system of ammonium oxidation differs from bacterial [7]. Activity of denitrification was estimated using traditional method measuring N2O in the presence of acetylene (10 kPa) [22]. Impact of thaumarchea in nitrous oxide emission from the soils compared to the control was estimated by the difference between the variants with the introduction of acetylene (10 Pa and 10 kPa). Gas samples were collected every 20 h. Rate of nitrous oxide accumulation in gaseous phase above soil samples was estimated using a 3700 gas chromatograph (Chromatograph, Russia). Length of a column packed with Polysorb–1 was 3.2 m, temperature was 40°C, and bridge current was 150 mA. Helium was used as carrier gas, and gas flow rates was 30 mL/min RESULTS AND DISCUSSION To check functional marker gene amoA primers, amplification was initially performed with pure culture of nitrifier Nitrosomonas europaea (Fig. 1, well 1). Additionally, PCR was conducted using DNA of Escherichia coli, which obviously lack this functional marker gene (Fig. 1, well 2), and without matrix DNA (negative control) (Fig. 1, well 3). Presence of characteristic band on agarose gel corresponding to N. europaea confirmed that PCR conditions were appropriate for AOB detection. Results of PCR analysis of functional marker gene amoA are shown (Fig. 1, well 5). Presence of characteristic bands on agarose gel corresponding to Nitrosomonas europaea and enrichment revealed that used PCR conditions were appropriate for AOB detection in soil samples. Results of PCR analysis of functional marker gene amoA of DNA extracted from soil samples are shown in Fig. 2 (wells 1 and 2). Pure culture of N. europaea was also used as a positive control and marker (Fig. 2, well 3), E. coli was used to check specificity of AmoA primers (Fig. 2, well 4), and water was used as negative control (Fig. 2, well 5). Bands of PCR products on electrophoregram on lanes 1, 2, and 3 corresponded to 320 bp, which confirmed the presence of nitrifiers in both soil samples. Bands were absent on lanes 4 and 5, since functional marker gene amoA was absent in E. coli and water. Specific band of sod-podzolic soil sample from agrocenose was more intensive than that from forest phytocenose that could indicate greater number in the soil of agrocenose. Quantitative analysis of the bands using ImageJ software revealed that index of abundance for this population was approximately 1.3–1.5 times higher than that for the forest soil. Thus, conditions for DNA extraction from soil samples and PCR amplification of fragments of functional marker gene of nitrification (amoA) used in the present study could be applied for detection of auto-
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1
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3 MR
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2
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4
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Fig. 2. Results of PCR-amplification of AmoA gene fragments of DNA samples extracted from (1, 2) sod-podzolic soil, (3) Nitrosomonas europaea, (4) Escherihea coli, (5) without matrix DNA (negative control); results of PCR-amplification of fragments of 16S rRNA genes extracted from sod-podzolic soil (6 is forest, 7 is agrocenose), (8) E. coli, (9) without matrix DNA (negative control); MR—DNA molecular weight marker (1000 bp).
4
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oxidizing bacteria was anticipated), formation of nitrous oxide was not detected. In the control experiment without ammonium and with acetylene (10 Pa), nitrous oxide was also not formed. Under these conditions, nitrification was inhibited and denitrifies did not developed due to the absence of enough amount of nitrate (for short duration of the experiment). Activity of thaumarchaea in this variant can be associated with archaea belonging to S cluster (soil archaea) that actively develop in the presence of ammonium.
7 MR
Fig. 1. Results of PCR-amplification of AmoA gene fragments of DNA samples extracted from: (1) Nitrosomonas europaea, (2) Escherihea coli, (3) without matrix DNA (negative control), (4) N. europaea, (5) enrichment (sodpodzolic soil, soil), (6) E. coli, (7) without matrix DNA (negative control); MR—DNA molecular weight marker (1000 bp).
trophic DNA in soil without traditional cultivation methods. Nitrogen fertilizers were regularly introduced in sod-podzolic soil, which resulted in increase of nitrification intensity and share of autotrophic nitrifiers in microbial community of agrocenose. This conclusion on features of ecosystems confirmed results of previous investigations [1, 4, 10, 15]. Studying of nitrification activity in the experiments with enrichments revealed that intense staining with Griess reagent after 7 d of cultivation was observed only in the enrichment from the arable. This result indicated the high activity of autotrophic nitrifiers in agrocenoses due to introduction of nitrogen fertilizers [18, 19]. Even after 2 weeks of incubation, enrichment from the forest phytocenose did not have red color, and slight color was detected only after 3 weeks of incubation. Thus, obtained results demonstrated that nitrifiers in the forest phytocenose had relatively low activity in comparison to agrocenose, where number and activity of autotrophic nitrifiers significantly exceeded those in the forest soil [11]. Estimation of the impact of thaumarchaea in gaseous losses of nitrogen from sod-podzolic soil in the form of N2O demonstrated that nitrous oxide was revealed only in microcosms with ammonium and acetylene (10 Pa) (Table 2), i.e., only archaeal nitrification was detected. This confirmed data obtained by other researchers [12]. In the variants with ammonium and without acetylene (where activity of ammoniaMOSCOW UNIVERSITY SOIL SCIENCE BULLETIN
In gray forest soils in the absence of acetylene, emission of nitrous oxide was not observed despite addition of ammonium sulfate inducing activity of ammonium-oxidizing bacteria. Introduction of acetylene in the gaseous phase (10 kPa) allowing us to estimate the impact of denitrifies shows low activity of denitrification due to low content of nitrates in the studied samples. Simultaneous introduction of acetylene (10 Pa) and ammonium resulted in increase of emission of nitrous oxide from the soil that could be caused only by activity of ammonia-oxidizing bacteria. Impact of ammonia-oxidizing archaea (AOA) in N2O emission from gray forest soil was calculated as Table 2. Dynamic of nitrous oxide emission from soil samples in presence of ammonium sulfate and acetylene Sod-podzolic soil, nM N2O/g 1 period 2 period 3 period Gray forest soil, nM N2O/g 1 period 2 period 3 period
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(NH)4SO4
(NH)4SO4 + C2H2
background level same ″
background level 784.9 1456.3
(NH)4SO4
(NH)4SO4 + C2H2
background level same ″
background level 477.576 1083.39
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the difference between activity of denitrifying bacteria and emission of nitrous oxide in the variant with introduction of ammonium and acetylene to inhibit ammonia-oxidizing bacteria (10 kPa). It comprised approximately 20–25% of total emission of nitrous oxide from this soil. CONCLUSIONS Results of PCR-amplification of fragments of bacterial and archaeal amoA genes confirmed results of previous studies, which demonstrated predominance of archaeal genes over bacterial in undisturbed soil and relatively high index of abundance of ammonia-oxidizing bacteria in agrocenoses at circum-neutral pH [6, 9, 12]. Impact of thaumarchaea in emission of nitrous oxide from gray forest soil could reach 20 to 25%. ACKNOWLEDGEMENTS The work was supported by the Russian Foundation for Basic Research, project no. 14-04-01573, and the Russian Academy of Sciences Presidium, project no. 14-26-00079. REFERENCES 1. Stepanov, A.L. and Lebedeva, E.V., Obrazovanie i pogloshchenie parnikovykh gazov v pochvennykh agregatakh (Greenhous Gases Formation and Adsorption in Soil Aggregates), Moscow, 2008. 2. Stepanov, A.L. and Lysak, L.V., Metody gazovoi khromatografii v pochvennoi mikrobiologii (Gas Chromatography Methods for Soils Microbiology), Moscow: MAKS Press, 2002. 3. Brochier-Armanet, C., Boussau, B., Gribaldo, S., and Forterre, P., Mesophilic Crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota, Natl. Rev. Microbiol., 2008, vol. 6, no. 3, pp. 245–252. 4. Chu, H., Fujii, T., Morimoto, S., et al., Community structure of ammonia-oxidizing bacteria under longterm application of mineral fertilizer and organic manure in a sandy loam soil, Appl. Environ. Microbiol., 2007, vol. 73, pp. 485–491. 5. Francis, C., Roberts, K., Beman, J., et al., Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean, Proc. Natl. Acad. Sci. U.S.A., 2005, vol. 102, pp. 14683–14688. 6. He, J.Z., Shen, J.P., Zhang, L.M., et al., Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices, Environ. Microbiol., 2007, vol. 9, no. 2, pp. 2364–2374. 7. Jia, Z. and Conrad, R., Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil, Environ. Microbiol., 2009, vol. 11, no. 7, pp. 1658–1671. 8. Könneke, M., Bernhard, A.E., de la Torre, J.R., et al., Isolation of an autotrophic ammonia-oxidizing marine archaeon, Nature, 2005, vol. 437, pp. 543–546.
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