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The role of mesophilic crenarchaea in soil nitrification

Final Report Summary - CRENECO (The role of mesophilic crenarchaea in soil nitrification)

Nitrification is a global biogeochemical process carried out by a range of microorganisms that, together, catalyse the aerobic oxidation of ammonia to nitrate. Nitrification proceeds in two steps, each catalysed by a specific subset of nitrifying microorganisms. The first step in the nitrification process is the oxidation of ammonia to nitrite by the aerobic ammonia oxidisers and the second to the oxidation of nitrite to nitrate by the aerobic nitrite oxidisers. Soil nitrification is responsible for considerable loss of ammonia-based fertilisers applied to agricultural fields at an annual cost of approx. USD 15.9 billion. The loss of ammonia-based fertilisers occurs through leaching of nitrate from soils, which results in groundwater pollution, and through emission of a potent greenhouse gas, nitrous oxide. Nitrous oxide has a global warming potential 298-fold greater than that of carbon dioxide given a time horizon of 100 years and is expected to become the largest cause of ozone depletion in the 21st century.

The oxidation of ammonia to nitrite is the rate-limiting step in the nitrification process and is therefore crucial. Autotrophic ammonia-oxidising bacteria were thought to be solely responsible for most of the ammonia oxidation in soil until the recent discovery of autotrophic, ammonia-oxidising mesophilic crenarchaea, now renamed thaumarchaea. Cultivated ammonia oxidising archaea grow with ammonia as sole energy source and assimilate inorganic carbon (carbon dioxide) as sole carbon source (autotrophy). Ammonia oxidising archaea have been cultivated from marine and thermal spring environments but not from soil. Indirect evidence, based on the detection and quantification of archaeal genes (amoA) involved in nitrification, suggests that putative archaeal ammonia oxidisers outnumber bacterial ammonia oxidisers in a wide range of soils and therefore have an important role in soil nitrification. The contribution of thaumarchaea to global carbon cycling and their mode(s) of carbon metabolism are poorly understood. Available studies suggested both autotrophy and heterotrophy as possible routes of carbon assimilation based on genomic and isotopic tracer studies. The aims of the CRENECO project were to assess the relative contribution of archaeal and bacterial ammonia oxidisers to soil ammonia oxidation and to determine whether autotrophic ammonia oxidation is a common trait of soil thaumarchaea.

The relative contribution of archaeal and bacterial ammonia oxidisers to soil nitrification was investigated by analysing their growth during active nitrification in soil microcosms incubated for 30 days at 30 degrees Celsius, and the effect of a known inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Soil microcosms consisted of 10 g of soil in 144-mL sterile serum bottles. Soil was collected from an agricultural plot (Scottish Agricultural College, Craibstone, Aberdeen) that has been maintained since 1961 at a pH of approximately 7. Community structure and growth of bacterial and archaeal ammonia oxidisers was assessed by DNA-based fingerprinting and quantification of 16S rRNA and amoA genes. Nitrification occurred in control microcosms as evidenced by a steady production of nitrite + nitrate. As no ammonia was added to the microcosms, the nitrification process was presumed to be supported by ammonia released by mineralisation of nitrogen-containing organic matter. No change in the composition and size of bacterial ammonia oxidiser community could be detected during the incubation of both control and acetylene-containing microcosms. In contrast, fingerprinting of archaeal 16S rRNA and amoA genes showed increases in the relative abundance of specific thaumarchaeal phylotypes during nitrification, suggesting growth. Growth of two thaumarchaeal phylotypes was demonstrated by increases in the abundance of corresponding amoA genes during incubation of control microcosms. Nitrification and growth of thaumarchaeal phylotypes were suppressed in microcosms containing acetylene, indicating that these archaea are ammonia oxidisers. This study demonstrated growth of archaeal but not bacterial ammonia oxidisers during active nitrification in a soil microcosm, providing compelling evidence that ammonia oxidation was mostly due to archaea in the conditions of our study.

The contribution of autotrophic carbon assimilation to growth strategies of thaumarchaea was assessed by studying the diversity and abundance of a thaumarchaeal autotrophy marker gene in soil and sediment samples. This required choosing an appropriate autotrophy marker gene and designing adequate PCR primers. The gene (hcd) encoding 4-hydroxybutyryl-CoA dehydratase, a key enzyme in the putative autotrophy pathway of the sequenced thaumarchaea Cenarchaeum symbiosum and Nitrosopumilus maritimus, was chosen as it is not known, so far, in heterotrophic archaeal lineages. PCR amplification products were obtained with newly designed PCR primers from two different agricultural plots (pH 7 and pH 4.5 respectively) from the long term field experiment in Craibstone, sediments sampled at two different locations of the Ythan estuary on the east coast of Scotland and deep-sea sediments collected from an abyssal plain (3,953 m depth) in the Pacific Ocean. Phylogenetic analysis confirmed that the newly designed primer pairs specifically target thaumarchaeal hcd genes and indicated the existence of environment-specific sequence clusters. The abundance of hcd genes in soil was 105 - 106 g-1 soil, as assessed by quantitative PCR. These preliminary data indicate that putative thaumarchaeal autotrophs are diverse, present in contrasting environments and abundant in soil. Community structure and growth of putative thaumarchaeal autotrophs was also characterised in the soil microcosms described above by fingerprinting and quantification of hcd genes. Nitrification in control microcosms coincided with increases in relative abundance of two hcd phylotypes and hcd gene abundance indicating growth of putative thaumarchaeal autotrophs during active nitrification. Nitrification and growth of the putative autotrophs was suppressed in acetylene-containing microcosms, providing evidence for autotrophic archaeal nitrification.

Strong evidence for autotrophic archaeal ammonia oxidation in soil was obtained by use of stable isotope probing to demonstrate assimilation of 13C-labelled carbon dioxide by amoA and hcd-possessing thaumarchaea in soil microcosms. Microcosms were constructed and incubated as described above and supplemented with 5 % 13C-CO2. Carbon dioxide assimilation was assessed by tracing heavy carbon (13C) into thaumarchaeal DNA. Heavy (13C-containing) DNA in soil DNA extracts was separated from light (12C-containing) DNA using caesium chloride density gradient ultracentrifugation. Nitrification coincided with increases in thaumarchaeal amoA and hcd gene copy numbers and relative abundance of two thaumarchaeal amoA phylotypes, as previously reported. In contrast, the size and composition of the ammonia oxidising bacterial community remained unchanged during the incubation period. Thaumarchaeal amoA and hcd genes were detected in the same fractions of the density gradient, suggesting that amoA and hcd are both linked to the same thaumarchaeal genomes and were exclusively found in heavy DNA after 28 days of incubation, providing the first direct evidence for autotrophic archaeal ammonia oxidation in soil. Bacterial amoA genes were amplified only from light DNA fractions further indicating that nitrification is dominated by archaea in our experimental system.

In conclusion, the CRENECO project demonstrated the existence of autotrophic archaeal ammonia oxidation in soil, showed that soil ammonia oxidation is essentially an archaeal process in some instances and suggested that autotrophy is a common growth strategy of thaumarchaea. These results highlight the need to include autotrophic archaeal ammonia oxidation in current models of global nitrogen and carbon cycling in order to improve significantly the predictive power of these models. This will however require redefinition of the role of bacterial ammonia oxidisers in soil nitrification and identification of ecological niches of both ammonia oxidising thaumarchaea and bacteria. In this perspective, the relation between archaeal ammonia oxidation and organic matter mineralisation suggested by our results could be further tested. The central role of soil nitrification in global nitrogen cycling, nitrogen fertiliser loss, nitrate pollution of groundwater cause of stomach cancers and greenhouse gas emissions indicate that the CRENECO project and follow up research will impact on agriculture, environment quality and human health affairs, also on the fundamental understanding of global energy and matter fluxes in the geobiosphere and global warming.