Ammonia oxidation coupled to CO2 fixation by archaea and bacteria in an agricultural soil Jennifer Pratscher, Marc G. Dumont, and Ralf Conrad1 Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany Edited by James M. Tiedje, Center for Microbial Ecology, East Lansing, MI, and approved February 1, 2011 (received for review August 2, 2010) Ammonia oxidation is an essential part of the global nitrogen fixation, one should be able to identify the active autotrophic cycling and was long thought to be driven only by bacteria. Recent ammonia-oxidizing prokaryotes using SIP. So far, DNA-SIP findings expanded this pathway also to the archaea. However, analyses successfully showed autotrophy of ammonia-oxidizing most questions concerning the metabolism of ammonia-oxidizing bacteria in sediments of a lake (14) and an estuary (15) but failed to detect CO fixation of ammonia-oxidizing archaea in agri- archaea, such as ammonia oxidation and potential CO2 fixation, 2 remain open, especially for terrestrial environments. Here, we in- cultural soil (16), although potential activity of these archaea in vestigated the activity of ammonia-oxidizing archaea and bacteria soil has been reported before (17, 18). DNA-SIP of grassland soil in an agricultural soil by comparison of RNA- and DNA-stable iso- revealed autotrophic ammonia oxidation of archaea (19), but tope probing (SIP). RNA-SIP demonstrated a highly dynamic and exactly which groups of archaeal ammonia oxidizers contributed to this process remains unclear. Furthermore, the efficiency of diverse community involved in CO fixation and carbon assimila- 2 DNA-SIP depends solely on replication of cells, thus excluding tion coupled to ammonia oxidation. DNA-SIP showed growth of microorganisms that might be active but not growing. In this the ammonia-oxidizing bacteria but not of archaea. Furthermore, case, RNA-SIP (20) is assumed to yield more detailed in- the analysis of labeled RNA found transcripts of the archaeal formation regarding activity. acetyl-CoA/propionyl-CoA carboxylase (accA/pccB) to be expressed The aim of this study was to investigate CO2 assimilation and labeled. These findings strongly suggest that ammonia- linked to nitrification of ammonia-oxidizing prokaryotes in an fi oxidizing archaeal groups in soil autotrophically xCO2 using the agricultural soil using RNA-SIP and DNA-SIP in parallel. We 3-hydroxypropionate–4-hydroxybutyrate cycle, one of the two also wanted to detect expression of archaeal amoA by mRNA fi fi Crenarchaeota pathways recently identi ed for CO2 xation in . Cat- catalyzed reporter deposition (CARD)-FISH. Our findings pro- alyzed reporter deposition (CARD)-FISH targeting the gene encod- vide further evidence that the contribution of nitrifying archaea amoA ing subunit A of ammonia monooxygenase ( ) mRNA and 16S to ammonia oxidation and CO2 fixation in terrestrial environ- rRNA of archaea also revealed ammonia-oxidizing archaea to be ments might be substantial. numerically relevant among the archaea in this soil. Our results demonstrate a diverse and dynamic contribution of ammonia-oxi- Results dizing archaea in soil to nitrification and CO2 assimilation and that Nitrification Activity in SIP Incubations. For SIP, agricultural soil 13 their importance to the overall archaeal community might be larger microcosms were incubated with 5% C-labeled CO2 or un- 12 than previously thought. labeled C-CO2 for 12 wk. Concentrations of 1–5% CO2 are considered typical in soil (21). Weekly fertilization of the soil μ μ · −1 mmonia oxidation, the first step in nitrification, is crucial for with either 15 g or 100 g (NH4)2SO4-N g dryweight of soil Athe global nitrogen cycle. For a long time bacteria were (d.w.s.) resulted in stepwise production and increase of nitrate believed to be solely responsible for this process and exclusively (Fig. S1), whereas nitrate concentration in the unfertilized con- to possess the genes for the ammonia monooxygenase (amo), the trol did not increase. As expected, the largest nitrate production fi was observed in the microcosms fertilized with the higher con- key enzyme of nitri cation (1). Now there is increasing evidence μ · −1 that archaea also are involved. Archaeal genes encoding subunit centration of ammonia (100 gNg d.w.s.). Ammonium and A of ammonia monooxygenase (amoA) have been found to oc- nitrite did not accumulate over time, indicating that nitrate cur in a wide variety of environments including marine systems, production indeed resulted from ammonia oxidation. Ammonia – and nitrate concentrations were not balanced because net nitri- hot springs, and soils (2 5). Furthermore, molecular studies fi fi revealed that ammonia-oxidizing archaea often outnumber the cation generally underestimates gross nitri cation in soils be- nitrifying bacteria in most environments by orders of magnitude cause of additional nitrogen cycling (22). (3, 5, 6). These findings all demonstrate the potentially signifi- cant role of archaea in the process of nitrification. Evidence also RNA-SIP. For RNA-SIP of ammonia-oxidizing prokaryotes, buoyant density centrifugation was conducted with all RNA ex- suggests the assignment of the ammonia-oxidizing archaea 12 13 should be assigned to the archaeal phylum Thaumarchaeota in- tracts from C and C microcosms after 8 and 12 wk of in- stead of to the Crenarchaeota (7, 8). In addition, these archaea cubation. The quantitative distribution of archaeal and bacterial may be of importance for the global carbon cycle. The ammonia- amoA transcripts in these gradients was analyzed by quantitative oxidizing archaea isolated from aquatic environments were all PCR (qPCR) of cDNA (Fig. 1). The copy numbers obtained – represent mean results from the triplicate microcosms and re- shown to be autotrophs (9 11), like their bacterial counterparts, 13 and analysis of 13C-bicarbonate–labeled lipid biomarkers of peated qPCR analyses. After 8 wk of incubation with 5% CO2 natural Crenarchaeota in the North Sea indicated an autotrophic metabolism (12). However, it still is unclear whether ammonia- oxidizing archaea in soil also have an autotrophic metabolism Author contributions: J.P., M.G.D., and R.C. designed research; J.P. performed research; and to what extent they are functionally active. An answer to this J.P. analyzed data; and J.P. and R.C. wrote the paper. question and a link of phylogeny to function could be provided The authors declare no conflict of interest. by stable isotope probing (SIP) of nucleic acids. This technique This article is a PNAS Direct Submission. allows the specific identification of microorganisms assimilating Data deposition: The sequences reported in this paper have been deposited in the Gen- labeled substances, most commonly carbon from a particular Bank database (accession nos. HM996921–HM996934, HQ293120–HQ293148,and 13C-labeled substrate (13). Direct demonstration of ammonia HQ685759–HQ685837). oxidation by this method is not possible, because nitrite, the 1To whom correspondence should be addressed. E-mail: [email protected]. product of ammonia oxidation, is not assimilated. However, as- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. suming that ammonia oxidation is coupled to autotrophic CO2 1073/pnas.1010981108/-/DCSupplemental. 4170–4175 | PNAS | March 8, 2011 | vol. 108 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1010981108 Downloaded by guest on September 24, 2021 −1 12 and fertilization with 15 μgN·g d.w.s., the copy number of observed. Also, controls with CO2 and the unfertilized samples archaeal amoA transcripts already showed detectable labeling in did not show any labeling (Fig. S3). These results suggest that, − the heavy fraction (1.81–1.83 g·mL 1) (Fig. 1A). This clear la- although archaea were actively involved in ammonia oxidation beling could not be seen with the fertilization treatment with 100 and CO assimilation, as shown in the RNA-SIP approach, they − 2 μgN·g 1 d.w.s. (Fig. 1C). Instead, a shift of the archaeal amoA did not replicate. By contrast, the ammonia-oxidizing bacteria mRNA toward the partially labeled, intermediate gradient acquired heavy DNA to such an extent that those not pro- − fraction (1.79–1.80 g·mL 1)of13C gradients was observed. This liferating could no longer be detected in the light DNA fractions. result indicates that archaea might have been inhibited by the This notion was supported by a comparison of the copy numbers − elevated ammonia concentration in the 100 μgN·g 1 d.w.s. of archaeal and bacterial amoA genes in the initial soil versus treatment, resulting in a slower activation and lower activity of copy numbers in the incubated soil, determined by qPCR. Al- ammonia oxidation. This notion is supported by the results of 12- though ammonia-oxidizing bacteria showed strong growth within − wk incubation. Although the archaeal amoA transcripts in the 15 the 12 wk of incubation (copy number·g 1 d.w.s. : 5.47 ± 0.75 × − μgN·g 1 d.w.s. treatment were shifted almost completely into the 106 in initial soil and 7.80 ± 0.67 × 107 after 12-wk incubation), this − heavy fraction (Fig. 1B), the transcripts of the 100 μgN·g 1 d.w.s. growth could not be observed for the archaea containing amoA − fertilization were still labeled only partially (Fig. 1D). The copy (copy number·g 1 d.w.s. : 4.77 ± 0.51 × 107 in initial soil and 5.35 ± number of bacterial amoA transcripts also peaked after 8 wk of 0.39 × 107 after 12-wk incubation). 13 incubation in the fractions with heavy RNA from the CO2 treatment (Fig. 1 E and G). Here, the labeling was stronger with Phylogenetic Analyses of Archaeal 16S rRNA in RNA-SIP and of amoA − the 100 μgN·g 1 d.w.s. treatment (Fig. 1E) than in the micro- Transcripts in RNA- and DNA-SIP. The heavy and light RNA frac- − − cosms fertilized with 15 μgN·g 1 d.w.s.