IN / 31

See also: ; Emis- Powlson DS (1993) Understanding the . sions; Isotopes in Soil and Investigations; Soil Use and Management 9: 86–94. Nitrogen in Soils: Cycle; Nitrification; Plant Uptake; Powlson DS (1999) Fate of nitrogen from manufactured Symbiotic Fixation; Pollution: Groundwater in agriculture. In: Wilson WS, Ball AS, and Hinton RH (eds) Managing Risks of to Humans Further Reading and the Environment, pp. 42–57. Cambridge: Royal Society of Chemistry. Addiscott TM, Whitmore AP, and Powlson DS (1991) Powlson DS (1997) Integrating agricultural nutrient man- Farming, Fertilizers and the Problem. Wallingford: agement with environmental objectives – current state CAB International. and future prospects. Proceedings No. 402. York: The Benjamin N (2000) Nitrates in the human diet – good or Fertiliser Society. bad? Annales de Zootechnologie 49: 207–216. Powlson DS, Hart PBS, Poulton PR, Johnston AE, and Catt JA et al. (1998) Strategies to decrease nitrate Jenkinson DS (1986) Recovery of 15N-labelled in the Brimstone Farm experiment, Oxfordshire, UK, applied in autumn to winter wheat at four sites in eastern 1988–1993: the effects of winter cover crops and England. Journal of Agricultural Science, Cambridge unfertilized grass leys. Plant and Soil 203: 57–69. 107: 611–620. Cheney K (1990) Effect of nitrogen fertilizer rate on soil Recous S, Fresnau C, Faurie G, and Mary B (1988) The fate nitrate nitrogen content after harvesting winter wheat. of labelled 15N and nitrate applied to a Journal of Agricultural Science, Cambridge 114: winter wheat crop. Plant and Soil 112: 205–214. 171–176. Wilson WS, Ball AS, and Hinton RH (eds) (1999) Managing Davidson EA (1991) Fluxes of and nitric Risks of Nitrates to Humans and the Environment. oxide from terrestrial . In: Rogers JE and Cambridge: Royal Society of Chemistry. Whitman WB (eds) Microbial Production and Con- sumption of Greenhouse Gases: , Nitrogen Oxides, Halomethanes, pp. 219–235. Washington, DC: American Society for . Dykhuizen RS, Fraser A, Duncan C, Golden M, Benjamin Nitrification N, and Leifert C (1999) Antimicrobial effect of acidified on gut pathogens: the importance of dietary ni- J I Prosser, University of Aberdeen, Aberdeen, UK trate in host defences. In: Wilson WS, Ball AS, and ß 2005, Elsevier Ltd. All Rights Reserved. Hinton RH (eds) Managing Risks of Nitrates to Humans and the Environment, pp. 295–316. Cambridge: Royal Society of Chemistry. Introduction Goss MJ, Howse KR, Christian DG, Catt JA, and Pepper TJ (1998) Nitrate leaching: modifying the loss from miner- Nitrification is the oxidation of reduced forms of alized organic matter. European Journal of Soil Science nitrogen, ultimately to nitrate. It is carried out by 49: 649–659. three microbial groups: (1) autotrophic Goulding K (2000) Nitrate leaching from arable and oxidizers, (2) autotrophic nitrite oxidizers, and (3) het- horticultural land. Soil Use and Management 16: erotrophic nitrifiers. Autotrophic ammonia and ni- 145–151. trite oxidizers are characterized by their ability to Goulding KWT, Poulton PR, Webster CP, and Howe MT oxidize ammonia sequentially to nitrate under aerobic (2000) Nitrate leaching from the Broadbalk wheat conditions, but these organisms possess a wider meta- experiment, Rothamsted, UK, as influenced by fertilizer and manure inputs and weather. Soil Use and Manage- bolic diversity. This has significant consequences for ment 16: 244–250. their ecology, for the occurrence and kinetics of nitri- Hatch D, Goulding K, and Murphy D (2002) Nitrogen. fication, and for interactions with other organisms. In: Haygarth PM and Jarvis SC (eds) Agriculture, The major sources of reduced forms of soil nitrogen Hydrology and Water Quality, pp. 7–27. Wallingford: are excretion and decomposition of organic nitrogen, CAB International. derived from animals and , and application Jarvis SC (2000) Progress in studies of nitrate leaching from of ammonia-based fertilizers. Many grassland. Soil Use and Management 16: 152–156. and plants require ammonium for growth, while Macdonald AL, Powlson DS, Poulton PR, and Jenkinson others assimilate nitrate. For both groups, nitrifica- DS (1989) Unused fertilizer nitrogen in arable soils – its tion is important in regulating the supply or loss of contribution to nitrate leaching. Journal of the Science of nitrogen from the environment. While ammonia has a Food and Agriculture 46: 407–419. Macdonald AJ, Poulton PR, Powlson DS, and Jenkinson DS propensity to bind to soil particles, its conversion to (1997) Effects of season, soil type and cropping on re- nitrate leads to significant losses of soil nitrogen coveries, residues and losses of 15N-labelled fertilizer through leaching and conversion to gaseous forms applied to arable crops in spring. Journal of Agricultural through . Nitrification also contributes Science, Cambridge 129: 125–154. to soil acidification, with consequent increases in 32 NITROGEN IN SOILS/Nitrification mobilization of toxic metals, particularly in heavily In the first step, ammonia is oxidized to hydroxyl- fertilized and poorly buffered soils. In addition, nitri- amine by a membrane-bound ammonia monooxygen- fiers are involved in production and conversion of a ase. The reaction is endergonic and requires number of greenhouse gases. and a source of reducing equivalents. is then oxidized to nitrite by hydroxylamine oxido- Autotrophic Ammonia-Oxidizing reductase, using oxygen from water in an exergonic reaction. Two electrons are required for ammonia oxi- dation, while the remainder pass down the . The NOÀ/NH potential is Autotrophic ammonia oxidizers are Gram-negative 2 3 À340 mV and reduced nicotinamide adenine dinucleo- bacteria, traditionally placed within the - tide (NADH) is synthesized by reverse electron flow iaceae and characterized by their ability to oxidize using adenosine triphosphate (ATP). Hydrogen ions ammonia. Further classification is based on a limited generated by the second step lead to acidification of number of characteristics, including cell shape, unbuffered , limiting sustained growth. internal membrane structures, flagella, inclusion An alternative mechanism for the first step has bodies, salt tolerance, activity, and substrate recently been proposed, involving nitrogen dioxide affinity and inhibition. Confirmation of taxonomic and tetroxide, based on studies of N. eutropha: groupings and descriptions is by DNA:DNA þ À hybridization of pure cultures. The type strain, Nitro- NH3 þ N2O4 þ 2H þ 2e ! NH2OH þ 2NO þ H2O somonas europaea, has been the subject of the 2NO þ O2 ! 2NO2ðN2O4Þ majority of biochemical, physiological, and genomic studies. This mechanism does not appear to involve ammonia More recently, analysis of sequences of 16S riboso- monooxygenase, and studies have also demonstrated mal ribonucleic acid (rRNA) and amo A genes (encod- reduction in lag phase and the stimulation of growth, ing the active subunit of ) activity, and cell yield in cultures supplied with nitric has enabled phylogenetic classification of cultured oxide. Any reduction in lag phase will provide ammo- ammonia oxidizers. This places Nitrosococcus ocea- nia oxidizers with advantages in soil where ammonia nus in the - and other known auto- supply is intermittent. trophic ammonia oxidizers in seven lineages within a monophyletic group within the -proteobacteria: Nitrosospira, europaea/Nitrosococcus Organic carbon is obtained by nitrifiers via carbon mobilis, , Nitrosomonas ma- dioxide fixation, and all strains studied as of 2003 rina, , Nitrosomonas cryo- possess type I Rubisco genes, but with sequence vari- tolerans,andNitrosomonas sp. Nm143. Difficulties in ation between strains. Rubisco in some strains cultivating ammonia oxidizers limit available physio- are located in carboxysomes. generated by am- logical data, but links exist between these phylogene- monia oxidation is only just sufficient for growth, par- tic groups, their physiological characteristics, and ticularly as fixation requires significant their environmental origin. Analysis of 16S rRNA reducing equivalents, and generation of reducing gene sequences also indicates evolutionary relation- equivalents consumes ATP. ships with photosynthetic bacteria and other groups Ammonia monooxygenase has broad substrate possessing intracytoplasmic membrane structures, sug- specificity, and ammonia oxidizers can oxidize and gesting that ammonia oxidizers evolved from photo- assimilate, but not grow on, a number of organic com- synthetic organisms. 16S rRNA sequence analysis pounds, including , bromoethane, ethylene, suggests only peripheral associations with methane propylene, cyclohexane, , and phenol. Acet- oxidizers, but similarities exist between amoAgenes ate, formate, and pyruvate can increase yield of -proteobacterial ammonia oxidizers (N. oceanus), by as much as 130%, but higher concentrations in- and pMMO (particulate ), hibit growth. The ecological implications for this are suggesting a common ancestral origin. not clear, but assimilation of soil organic carbon Biochemistry may provide a selective advantage, while ammonia oxidizers may be important in biodegradation of or- Ammonia oxidation by -proteobacteria involves ganic pollutants in oligotrophic environments, where two steps: alternative carbon sources are scarce. Ammonia monooxygenase is similar to the mem- NH þ O þ 2Hþ þ 2eÀ ! NH OH þ H O 3 2 2 2 brane-bound methane monooxygenase of methano- À þ À NH2OH þ H2O ! NO2 þ 5H þ 4e trophic bacteria. Ammonia oxidizers can oxidize but NITROGEN IN SOILS/Nitrification 33

þ À À þ À not grow on methane and carbon monoxide, and the NH4 þ 1:32NO2 þ 0:066HCO3 þ 0:13H ! 0:26NO3 enzymes have common inhibitors. Incorporation of þ1:02N2 þ 0:066CH2O þ 0:5N0:15 þ 2:03H2O methane is inhibited by ammonia, and vice versa, and complete methane oxidation may be prevented by However, neither of these anaerobic processes has inhibition of hydroxylamine oxidation by formalde- been detected in soil, where their significance is un- hyde, produced during methane oxidation. In gen- clear. Both require substrates which are produced eral, ammonia oxidizers have higher oxidation rates under oxic conditions (nitrogen dioxide and nitrite) and lower Km values for ammonia than methano- but the processes may occur at oxic–anoxic inter- trophs, while the converse is true for oxidation and faces, such as in formed on soil particulate affinity for methane. Competition between the two material. groups for ammonia and methane therefore appears unlikely, but high levels of ammonia-based fertilizer may suppress activity. Despite these Autotrophic Nitrite-Oxidizing Bacteria considerations, ammonia oxidizer 16S rRNA and Nitrite oxidizers are classified into four genera within 13 amoA sequences have been found in C-labeled nu- the proteobacteria: Nitrobacter (the most studied; cleic acids extracted from grassland soils treated with -proteobacteria), Nitrococcus (-proteobacteria), 13 C-labeled methanol and methane, suggesting their (-proteobacteria), and Nitrospina (form- role in soil methane oxidation. Ammonia oxidizers ing its own subdivision). Nitrobacter strains are may also have a role in the oxidation of another related to several denitrifying organisms, while Nitro- greenhouse gas, carbon monoxide. They have greater bacter and Nitrococcus possess intracytoplasmic affinity for carbon monoxide than carboxydobac- membranes and are related to photosynthetic bac- teria, and may provide an important carbon monox- teria. -Proteobacterial nitrite oxidizers do not con- ide sink. Carbon monoxide oxidation only occurs tain intracytoplasmic membranes and are not related during ammonia oxidation, which is required as an to photosynthetic organisms. Only Nitrobacter and energy source, even though carbon monoxide and Nitrosospira have been detected in soil. Nitrite oxi- ammonia compete for the ammonia monooxygenase dation is catalyzed by , the À À . NO3 /NO3 redox potential is 430 mV, and reducing equivalents are generated by reverse electron flow, Ammonia Oxidation under Anaerobic Conditions consuming ATP: À À þ À Although traditionally considered to be aerobic, NO2 þ H2O ! NO3 þ 2H þ 2e autotrophic ammonia oxidizers maintain activity at low oxygen concentrations and under anaerobic con- Nitrite oxidizers fix CO2 for growth but, unlike ditions. N. europaea possesses a and ammonia oxidizers, many are capable of mixotrophic can denitrify nitrite under anaerobic conditions using and heterotrophic growth on a range of simple hydrogen or organic compounds as electron donors. organic substrates. Heterotrophic growth is slow (They can also denitrify under oxic conditions, using (doubling times of 30–150 h) but leads to greater ammonia as electron donor.) This results in the pro- biomass yields. Nitrite oxidoreductase enables het- duction of nitric and nitrous oxides, and dinitrogen erotrophic growth under anaerobic conditions, with gas. At atmospheric oxygen concentrations, nitrogen nitrate as the electron acceptor and production of oxides constitute 0.1–0.5% of the total N oxidized, nitrite and ammonia, and nitrogen oxides. This meta- but proportions rise to 2.5–10% at low oxygen bolic versatility may explain greater abundance of concentrations. nitrite oxidizers than ammonia oxidizers in soil. Anaerobic ammonia oxidation by N. eutropha has recently been demonstrated in which nitrogen Heterotrophic Nitrification dioxide or nitrogen tetroxide acts as an electron ac- ceptor, generating and hydroxylamine. Heterotrophic nitrification is the oxidation of inor- The latter is converted to nitrite with release of ganic and organic reduced forms of N, to nitrate, by a dinitrogen gas. In addition, anaerobic ammonia oxi- wide range of fungi and heterotrophic bacteria. In dation is carried out by autotrophic planctomycetes, some organisms, the mechanism is similar to that in which utilize nitrite as an electron acceptor, gener- autotrophic ammonia oxidizers and is linked, in some ating nitrate and nitrogen, with hydrazine and strains, to aerobic denitrification. The second mech- hydroxylamine as intermediates. The process is sensi- anism, termed fungal nitrification, is linked to lignin tive to oxygen and nitrite, and the calculated mass degradation and involves reaction of reduced organic balance is: compounds with hydroxyl radicals produced in the 34 NITROGEN IN SOILS/Nitrification presence of hydrogen peroxide and superoxide. There in grassland soils. Regular application of inorganic is little evidence that gain energy or nitrogen fertilizer reduces heterogeneity in ammonia other benefit from nitrification, and cellular rates of oxidizer communities, in association with similar heterotrophic nitrification activity are significantly reduction in the heterogeneity of soil pH and ammo- lower than for . Nevertheless, it may be nia concentration. Analysis of nitrification kinetics important in acid soils or where C:N ratios and during incubation is therefore important in providing biomass are high. information on soil population levels and substrate concentrations. Growth parameters are more easily obtained from laboratory cultures and typical values Analysis of Nitrifiers and Nitrification are given in Table 1. Other nitrogen cycle processes, e.g., denitrification, Nitrification Activity and Rates immobilization, and ammonification, will influence Nitrification rates in soil are traditionally measured concentrations of ammonium, nitrite, and nitrate. as ammonia consumption or nitrite plus nitrate Rigorous measurement of soil nitrification rates, and production during incubation of soil or soil slurry. distinction between autotrophic and heterotrophic Zero-order kinetics result from short-term measure- nitrification therefore necessitate the use of metabolic ments at nonlimiting ammonia concentrations and blocks and 15N-based techniques. The former involve measure potential nitrification, which can be used to measurement of nitrate production in the presence estimate nitrifier abundance. Short-term measure- of specific nitrification inhibitors e.g., nitrapyrin and 15 þ ments at limiting substrate concentrations generate . The second involves applying NasNH4 À Michaelis–Menten kinetics. Long-term incubation or NO3 and following changes in the concentration of nonenriched soil leads to exponential increases in of label in the respective pools. nitrite plus nitrate owing to unrestricted growth, at nonlimiting substrate concentrations, or growth at Enumeration submaximal and decreasing rates at limiting substrate concentrations. Nitrate-production kinetics are fre- Dilution-plate enumeration of viable nitrifiers is not quently fitted to the logistic equation. Kinetics can be feasible because of poor growth on solid media and interpreted differently, however, if nitrifiers are con- enumeration is traditionally achieved using the most sidered to be distributed heterogeneously, as clusters probable number (MPN) method. This involves mul- growing on discrete sources of ammonium within the tiple inoculation from dilutions of a soil suspension soil. If nitrification is measured in individual soil into mineral-salts medium containing ammonium aggregates, the observed nitrate production curves and a pH indicator. After incubation for several fit well with the cumulative distribution curves for (typically 4) weeks, growth is assessed by nitrite pro- lag periods. Bulk nitrate concentrations therefore re- duction, ammonium loss, and/or acidification, and flect not cumulative product of a uniformly growing cell concentrations are calculated using statistical population but the continued activation of clusters of tables. The requirement for laboratory cultivation . The increase in nitrate concentra- leads to underestimation of cell concentrations. In tion then depends on the distribution of lag periods addition, growth may be biased by ammonium con- prior to the onset of nitrification. Further evidence for centrations in the medium, with inhibition of some heterogeneous distribution of ammonia oxidizers in strains at high concentrations (Figure 1). MPN counts soil is provided by molecular analysis of communities for typical soils range between 103 and 105 cells gÀ1

Table 1 Reported ranges for kinetic constants derived from pure cultures of ammonia and nitrite oxidizers

Ammonia oxidizers Nitrite oxidizers

Maximum specific growth rate (hÀ1) 0.02–0.088 0.018–0.058 Doubling time (h) 8–35 12–39 Biomass yield (g biomass molÀ1 substrate) 0.42–1.72 1.11–1.51 Cell yield (cells molÀ1 substrate) 1.85–10.6 À À Carbon yield (ratio of CO2 fixed: NO2 or NO3 produced) 0.014–0.096 0.013–0.031 À À À1 À1 Cell activity (fmol NO2 or NO3 cell h ) 0.9–31.3 5.1–42 À À À1 À1 Biomass activity (fmol NO2 or NO3 g biomass h ) 4–200 15.7–25.2 À1 þ À Saturation constant for growth (mmol l NH4 or NO2 ) 0.12–14 0.045–0.178 À1 þ À Saturation constant for activity (mmol l NH4 or NO2 ) 0.05–0.07 1.6–3.6 NITROGEN IN SOILS/Nitrification 35

Figure 1 Enumeration of ammonia-oxidizer cells in long-term ecological research (LTER) soils using the most probable number þ À1 (MPN) method with medium containing 5, 50, or 1000 mg of NH4 -N ml and competitive polymerase chain reaction (C-PCR). Treatments are: Tr1, conventional tilling; Tr2, no tilling; Tr5, Populus perennial cover crop; Tr7, historically tilled; NDF, natural deciduous forest. Error bars represent the standard errors. Suffixes ‘T’ and ‘F’ indicate tillage and fertilization, respectively. (Reproduced with permission from Phillips CJ, Harris D, Dollhopf SL et al. (2000) Effects of agronomic treatments on the structure and function of ammonia oxidising communities. Applied and Environmental Microbiology 66: 5410–5418.)

soil for ammonia oxidizers, with nitrite oxidizers denaturing or temperature gradient gel electropho- slightly more abundant. Quantitative molecular tech- resis (DGGE, TGGE) and terminal restriction frag- niques (e.g., competitive PCR (cPCR), real-time PCR) ment length polymorphism (T-RFLP) analysis may avoid cultivation bias and detect the total nitrifier be used. These approaches do not require laboratory populations, giving cell concentrations 1–2 orders of cultivation and thereby eliminate many of the disad- magnitude greater than MPN counts (Figure 1). vantages of cultivation-based techniques, and difficul- ties in purifying and identifying ammonia-oxidizing Enrichment and Isolation bacteria. Autotrophic ammonia-oxidizing bacteria are readily The application of molecular techniques has enriched by inoculation of an inorganic medium, greatly expanded our knowledge of nitrifier commu- containing ammonium with soil, and incubation nities in soils. Phylogenetic analysis of amplified for several weeks. Isolation of pure cultures is more sequences reveals considerable diversity within soil difficult, due to faster growth of heterotrophs on or- ammonia oxidizers. Duplicate sequences and se- ganic carbon contaminants or by-products of nitrifier quences identical to pure cultures are rarely obtained, growth. This typically takes several months and cul- although this partly reflects the few pure culture se- turesfrequentlydonotsurvivecontinuedsubculturing. quences in databases and, potentially, sequencing and Laboratory selection also reduces the likelihood of cloning errors. Sequences are sometimes homologous obtaining dominant or representative environmental to those of laboratory cultures enriched from the strains, but enrichment of a strain is necessary for same environment and the majority fall within the unequivocal evidence of its presence and viability in lineages obtained with sequences from cultivated or- a particular soil. ganisms. One exception is the possible subdivision of Nitrosospira into four clusters, and there is evidence Soil Communities and Diversity of differences in physiological characteristics of pure Information on 16S rRNA and amoA gene sequences and enriched cultures representative of these different from cultured ammonia oxidizers has led to the design Nitrosospira clusters. of oligonucleotide polymerase chain reaction (PCR) Soils are dominated by -proteobacterial ammonia primers specific for -proteobacterial autotrophic oxidizers, with -proteobacterial sequences appar- ammonia oxidizers. These have been used to amplify ently restricted to marine environments. The links ammonia-oxidizer gene fragments from nucleic acids between 16S rRNA-defined lineages and physio- extracted directly from soil. Gene fragment sequences logical characteristics aid in the interpretation of the may then be analyzed by cloning, sequencing of distribution of these organisms under different soil clone library representatives, and phylogenetic analy- conditions. This approach also indicates that Nitro- sis. Alternatively, fingerprinting techniques such as sospira may be more abundant than Nitrosomonas in 36 NITROGEN IN SOILS/Nitrification soil. If so, more studies are required of Nitrosospira Influence of Soil Characteristics on physiology, rather than basing interpretation on Ammonia-Oxidizer Communities information on . Fingerprinting techniques, particularly DGGE, Soil pH have been used to study the effects of environmental Ionization of ammonia at low pH values reduces factors and soil treatments on ammonia-oxidizer substrate availability for ammonia oxidizers, and la- communities. This provides more rapid assessment boratory batch growth of ammonia oxidizers does and greater replication than cloning and/or sequenc- not occur at pH values of less than 6.5. However, ing approaches. In addition, bands of interest on autotrophic ammonia oxidizers with neutral pH DGGE gels may be excised, cloned, and sequenced optima are frequently isolated from acid soils, and and information on identity obtained by compari- autotrophic nitrification has been demonstrated in son with database sequences. These approaches soils with pH values as low as 3.5. In some situations, can also be used to assess quantitative or semiquanti- nitrification in acid soils is due to heterotrophic tative changes in ammonia-oxidizer community nitrification. Coupled denitrification–nitrification structure. by heterotrophic nitrifiers is unlikely, due to the re- Comparison of 16S rRNA gene sequences in en- quirement for high substrate concentrations, and richment cultures with those amplified from nucleic heterotrophic nitrification involving ammonia mono- acids extracted from the same soil provides an indi- oxygenase-based reactions suffers from reduced cation of selection during laboratory cultivation. ammonia availability at low pH. However,acidophilic The relative abundances obtained using these two fungal heterotrophic nitrifiers have been character- approaches differ but, in one study, 16% of sequences ized and fungal nitrification is significant in some were common to both enrichment cultures and clones acid soils. Nevertheless, use of specific inhibitors of analyzed. Molecular analysis of cultures obtained autotrophic ammonia oxidation and 15N-based stud- during MPN enumeration also indicates selection, ies have demonstrated active, autotrophic ammonia with frequent domination by Nitrosomonas strains oxidation in acid soils. in samples from environments which appear, from One explanation for this phenomenon is the pos- molecular analysis, to be dominated by Nitrosospira. session by some ammonia oxidizers of urease activity, Molecular- and cultivation-based techniques there- enabling growth on urea as a sole nitrogen source. fore provide different views of diversity and species Urease activity and growth on urea are independent of composition within soil ammonia-oxidizer com- pHintherange4–8,possiblythroughuptakebypassive munities. However, molecular techniques are also diffusion. Laboratory studies show that, under these subject to bias and additional techniques are re- conditions, ammonia oxidation can proceed at pH quired. In this respect, direct detection of cells using values as low as 4, until urea is exhausted, after which fluorescent in situ hybridization (FISH) with specific it ceases, even if ammonium is present in solution. ammonia-oxidizer probes would be useful. This This has been demonstrated in a strain of Nitrosospira approach, however, is limited by autofluorescence that cannot oxidize ammonium at pH values less and the relatively low cell concentrations of ammonia than 7 (Figure 2). Urease activity therefore provides oxidizers in soil.

Figure 2 Changes in pH and in concentrations of urea, ammonium, and nitrite during growth of Nitrosospira strain NPAV in liquid batch culture on poorly buffered medium containing urea, at initial pH values of (a) 4 and (b) 7. (Reproduced with permission from Burton SAQ and Prosser JI (2001) Autotrophic ammonia oxidation at low pH through urea hydrolysis. Applied and Environmental Microbiology 67: 2952–2957.) NITROGEN IN SOILS/Nitrification 37 the potential for acidophilic ammonia oxidation, but is not a prerequisite for ammonia oxidizers isolated from acid soils. Autotrophic ammonia oxidizers appear to be protected from low pH in soil when occurring at high cell concentrations and associated with par- ticulate matter. High cell concentrations can be achieved in aggregates, which when inoculated into liquid medium can nitrify at low pH, and nitrification at pH 4 has been demonstrated for cells immobilized in alginate. formation on surfaces such as glass slides, glass beads, ion-exchange resin beads, and clay minerals can reduce the pH minimum for growth and activity of N. europaea by up to 2 pH Figure 3 The influence of soil pH on the relative abundances of units. Surface growth protection increases with the Nitrosospira clusters 2 (CL2), 3 (CL3), and 4 (CL4), and Nitrosomo- cation exchange capacity of the particulate material nas cluster 6a. Abundances are calculated as relative intensities and lag phases prior to growth of stationary-phase or of denaturing gradient gel electrophoresis (DGGE) analysis of starved cells are reduced for attached cells. Protection ribosomal ribonucleic acid (rRNA) genes amplified from DNA extracted from soil maintained at a range of pH values, using from low pH may result from preferential growth ammonia oxidizer-specific primers. (Reproduced with permis- and nitrification at the clay surface, due to adsorp- sion from Stephen JR, KowalchukGA, Bruns MA et al. (1998) tion of ammonium. It is greatest when the only source Analysis of -subgroup ammonia oxidiser populations in soil by of ammonium is that bound to clay minerals. This has DGGE analysis and hierarchical phylogenetic probing. Applied been demonstrated by growth on ammonia-treated and Environmental Microbiology 64: 2958–2965.) vermiculite (ATV), in which ammonia is fixed to the vermiculite by high-temperature exposure to am- monia. In the presence of ATV, N. europaea growth The development of molecular techniques has occurs without a lag phase and with greater nitrite enabled assessment of the influence of soil pH on yield than for suspended cells, but with no decrease ammonia-oxidizer communities. In one study, DGGE in the pH of the medium. This, and the other clay analysis was carried out on -proteobacterial 16S effects, suggests localized buffering; ammonium re- rRNA gene fragments amplified from DNA extracted leased from the clay surface is utilized by adsorbed from soils maintained for 36 years at pH values cells and Hþ ions are effectively inactivated by ex- in the range 3.9–6.6. Relative abundances of differ- change for ammonium ions. It is not clear why ent clusters were assessed as relative intensities of biofilm growth should reduce the lag phase, but DGGE bands and showed a greater proportion of this presents a significant ecologic advantage in Nitrosospira cluster-2 sequences in acid soils and a competing for transient supplies of ammonium. greater proportion of Nitrosospira cluster 3 at higher A possible mechanism involves cell–cell signaling pH (Figure 3). Other clusters detected were Nitrosos- molecules, acyl homoserine lactones, which will ac- pira cluster 4, which may have increased in relative cumulate at high cell concentrations. When N. euro- abundance at pH 5.5, and Nitrosomonas cluster 6a, paea biofilms established on glass beads are starved which was independent of pH. Molecular studies of and then resupplied with ammonia, oxidation occurs agricultural, grassland, and forest soils provide fur- immediately. However, supply of ammonia to sus- ther evidence for the association of Nitrosospira clus- pended, nonbiofilm cells in liquid medium results ter-2 sequences with acid soils. However, lack of in a lag period, prior to ammonia oxidation. The information on distinctive physiological characteris- lag period increases with the length of the preceding tics specific to representatives of this group, and of starvation period but is significantly reduced by physiological diversity within ammonia oxidizers in addition of acyl homoserine lactones. Signaling general, prevents establishment of links between spe- compounds are produced by a wide range of soil cies diversity, functional diversity, and mechanisms microorganisms and affect a number of ecologically for nitrification at low pH. important processes. These compounds may there- fore mediate interactions between nitrifiers and Nitrogen Fertilization other soil microorganisms, particularly in biofilms and in the rhizosphere, where biomass concentrations Sustained nitrogen fertilization and liming increase are sufficient to lead to high concentrations of nitrifier population size, as indicated by cell signaling molecules. counts (MPN and cPCR) and nitrification potential 38 NITROGEN IN SOILS/Nitrification measurements. In addition, evidence from molecular Summary studies of nitrifiers in long-term ecological research Nitrifying bacteria convert the most reduced form (LTER) sites subjected to a number of different fertil- of soil nitrogen, ammonia, into its most oxidized ization and cultivation conditions indicates an influ- form, nitrate. In itself, this is important for soil eco- ence on the ammonia-oxidizer communities. Tilled system function, in controlling losses of soil nitro- soils were dominated by Nitrosospira cluster-3 se- gen through leaching and denitrification of nitrate. quences, while never-tilled soils contained Nitrosos- Nitrifiers also contribute to other important pro- pira clusters 3 and 4, and Nitrosomonas cluster 6. cesses, including nitrous oxide production, methane Similar effects of fertilization on the relative propor- tions of Nitrosospira cluster-3 and -4 sequences have oxidation, degradation of organic compounds, and carbon monoxide oxidation. The development of been found in chalk grassland soil. Little is known 15N-based techniques has increased significantly our about the persistence of changes after perturbations. ability to dissect soil nitrogen transformations and Nitrogen supplied as inorganic fertilizer or, in their rates, while molecular techniques now enable grazing systems, as animal urine leads to localized, characterization of soil nitrifier community structure high ammonia concentrations. These concentrations and changes in species composition. These ap- are sufficient to inhibit ammonia-sensitive groups of proaches are increasing our understanding of the ammonia oxidizers, while permitting growth of more tolerant strains. Soil microcosm experiments have ecology of soil-nitrifying bacteria and provide the potential for determining relationships between di- demonstrated that the kinetics of nitrification of am- versity, community structure, and function monia derived from synthetic sheep urine applied to in this important group of organisms. grassland soil depends on the relative abundances of ammonia-sensitive and ammonia-tolerant strains. Low numbers of the latter result in significant lag See also: Nitrogen in Soils: Cycle; Nitrates; Plant periods before nitrification, impact significantly on Uptake maintenance of high levels of ammonia for plant growth, and reduce nitrogen losses associated with leaching and denitrification of nitrate. Further Reading Belser LW (1979) Population ecology of nitrifying bacteria. Inhibition of Soil Nitrification Annual Review of Microbiology 33: 309–333. De Boer W and Kowalchuk GA (2000) Nitrification in acid Specific inhibitors of autotrophic nitrification (e.g., soils: microorganisms and mechanisms. and nitrapyrin, dicyandiamide, etridiazole) have been used Biochemistry 33: 853–866. in the field to reduce losses of fertilizer nitrogen and Koops H-P and Pommerening-Ro¨ ser A (2001) Distri- to reduce nitrate pollution. Inhibitors, particularly bution and ecophysiology of the nitrifying bacteria acetylene, are also used as metabolic blocks when emphasizing cultured species. FEMS Microbiology measuring nitrification rates. Higher concentrations Ecology 37: 1–9. of these compounds are required for inhibition in soil Koops H-P, Purkhold U, Pommerening-Ro¨ ser A, than in laboratory culture because of inactivation and Timmermann G, and Wagner M (2003) The lithoauto- trophic ammonia-oxidizing bacteria. In: Dworkin D, degradation and because attachment of nitrifiers to Falkow S, Rosenberg E, Schleifer K-H, and Stackebrandt soil surfaces protects cells from inhibition. Protection E (eds) The : An Evolving Electronic is greater with expanding clays with high cation ex- Resource for the Microbiological Community. 3rd edn, change capacity. In many mature coniferous soils, release 3.13. New York: Springer-Verlag. nitrogen is present as ammonium rather than nitrate Kowalchuk GA and Stephen JR (2001) Ammonia-oxidizing and net nitrification rates are low. This has led to sug- bacteria: a model for molecular microbial ecology. gestions of allelopathic inhibition of nitrification by Annual Review of Microbiology 55: 485–529. polyphenolic compounds released by roots, which Laanbroek HJ and Woldendorp JW (1995) Activity of can inhibit laboratory growth of nitrifiers. However, chemolithotrophic nitrifying bacteria under stress in use of 15N-based techniques has demonstrated high natural soils. Advances in Microbial Ecology 1: gross nitrification rates in these soils and low nitrate 275–304. Prosser JI (ed.) (1986) Nitrification. Oxford, UK: IRL Press. levels are more likely due to greater immobilization of Prosser JI (1989) Autotrophic nitrification in bacteria. nitrate. There is little direct evidence for specific Advances in Microbial Physiology 30: 125–181. effects of plant diversity and community structure Prosser JI and Embley TM (2002) Cultivation-based and on ammonia-oxidizer communities, although plants molecular approaches to characterisation of terrestrial can exert indirect effects through their influence on and aquatic nitrifiers. Antonie van Leeuwenhoek 81: soil ammonium concentrations. 165–179. NITROGEN IN SOILS/Plant Uptake 39

Prosser JI, Embley TM, and Webster G (2002) The influ- with rhizobium and Frankia bacteria, respectively. ence of selection pressures on , func- These symbionts form nodules on the roots which tional gene diversity and activity of ammonia oxidising are capable of fixing dinitrogen and thus supply bacteria. In: Hails R, Beringer JE, and Godfray HCJ the plant with N directly. The majority of plants, (eds) Genes in the Environment, pp. 187–202. Oxford, however, do not form N -fixing symbiotic associ- UK: Blackwell Publishing. 2 ations and must rely on N uptake via their roots or Schmidt I, Slickers O, Schimid M et al. (2002) Aerobic and anaerobic ammonia oxidizing bacteria – competi- mycorrhizas. tors or natural partners? FEMS Microbiology Ecology Most transformations of N also depend on the 39: 175–181. supply of carbon (C), and thus these two nutrient cycles are closely linked. The C:N ratio of the sub- strate determines the rates at which N (and C) are released and mineral N made available for plant uptake. Also influential are the composition of the Plant Uptake decomposer microbial community and its require- A Hodge, University of York, York, UK ments for N. Fungi tend to have a higher substrate assimilation efficiency and a lower N requirement per ß 2005, Elsevier Ltd. All Rights Reserved. unit of C assimilated than bacteria. Thus, if the de- composer population is predominantly fungal, more Introduction N may be released than if it was predominantly bac- terial. However, fungi also tend to turn over less Nitrogen (N) is essential for life because it is a com- rapidly than bacterial populations, so the N contained ponent of nucleic acids and amino acids and thus in their structural components will be locked up for a peptides and proteins. In addition, plants also require longer time than bacterial N. Generally net mineral- N for chlorophyll. In most terrestrial ecosystems, N is ization, resulting in the release of N, occurs if the C:N the primary limiting nutrient. In soils, N is present ratio of the substrate being decomposed is lower than predominantly in organic form, and inorganic N is approx. 25:1. At higher C:N ratios, net immobiliza- made available by the mineralization of organic N to tion occurs and the microbial decomposer commu- þ ammonium (NH4 ) and subsequent oxidation to ni- nity requires additional N from the soil system to À trate (NO3 ). Both these inorganic N sources can be decompose the substrate. taken up by plant roots, but roots have to compete Net immobilization of N reduces the N available with soil microorganisms as well as other plant roots for the plant and can reduce overall plant growth. for this N. Recent evidence suggests that plants may Although other factors influence the release of N, be able to access simple organic N sources directly for example lignin content of the substrate, there is without the need for the mineralization process to a strong inverse relationship between the amount of release inorganic N, but these N sources also have plant N captured from organic substrates and the to be captured in a competitive environment. The C:N ratio of the substrate, as shown in Table 1. distribution of N sources in soil, particularly organic In addition to microorganisms soil animals also N forms, is both spatially and temporally heteroge- play an important role in the mineralization process neous. Plant roots respond to this heterogeneity in a through physical, chemical, and biological processes. number of ways in order to capture N from the soil Soil animals can physically break up and redistribute system. organic materials in the soil. They also ingest organic materials which are subsequently broken down by a Plant–Microbial Competition for N range of enzymes and/or by microflora present in their guts. Much of the C they consume is used for meta- þ Mineralization of organic material to NH4 is carried bolic processes and the excreted waste products, in þ out by a wide range of soil animals and microorgan- the form of NH4 , urea, or amino acids, are a much isms. However, mineralization and the subsequent more readily available form of N for plant uptake than þ À conversion of NH4 to NO3 are generally considered the organic material originally ingested. Soil animals to be the rate-limiting steps in the terrestrial N-cycle. feed on microorganisms as well as other soil animals. Due to their rapid turnover times and rapid The plant benefits from the regulation of the size and adaptation rate, microorganisms are generally activity of the microbial population, and from the thought to utilize inorganic N sources first, with the release of more N into the soil–plant system. An plant only being able to access what is left over after increase in plant N capture of up to 75% has been microbial uptake. Exceptions are plants such as reported as a result of protozoa grazing on soil mi- legumes and alder which form symbiotic associations croorganisms. Thus, although microorganisms may