Ecology of Nitrogen Fixing, Nitrifying, and Denitrifying Microorganisms in Tropical Forest Soils

1Nitrogen (N) is an important component of proteins and nucleic acids, and thus is an essential nutrient for all organisms. Furthermore, the N supply to organisms regulates primary productivity in many natural ecosystems. N exists in multiple oxidation states and chemical forms in nature, and transformations among these different forms are typically catalyzed by microorganisms. Soil microorganisms constitute a major portion of the biodiversity in soils and play a pivotal role in soil processes, which ultimately affect the functioning of terrestrial ecosystems. Given the biogeochemical contributions of microbes, microbial community dynamics may be associated with temporal and spatial variation in N processes and rates. Thus, understanding the link between biogeochemical N processes and microbial community dynamics can provide a more mechanistic understanding of the N cycle than the direct observation of N dynamics. Likewise, it is important to know the factors that influence the biodiversity and functioning of soil microbes that mediate these processes in order to predict ecosystem responses to a changing environment.

2Tropical forests are particularly important in the N cycle. Although these ecosystems occupy only 12% of the Earth’s surface, they contribute ∼70% of terrestrial N fixation and emit ∼50% of nitrous oxide (N2O; ). They are a major reservoir of biodiversity and play a vital role in regulating the Earth’s climate and biogeochemical cycles through their vast exchanges of energy, water, and nutrients with the atmosphere. Tropical forests account for nearly 40% of terrestrial net primary production, contain 25% of the world’s biomass carbon (C) and play a critical role in buffering the atmosphere against rising carbon dioxide (CO2). On the other hand, these forest ecosystems are threatened by high levels of deforestation, rapid rates of land conversion to agriculture, and chronic-elevated N deposition, with important implications for global climate. As a result, there have been increased efforts recently to understand and predict how soil microbial communities in tropical forests regulate biogeochemical processes, in particular N cycling.

3Our understanding of biological N cycling in forest ecosystems has undergone a major shift in the past decade in concert with the growing interest in integrating evolutionary and ecological theory into microbial ecology, the rapid development of molecular-based culture-independent methods, and the use of N isotope tracer techniques. These advances have increased our understanding of microbial communities, their functional potential, and their physiological state, and have sparked a new interest in the functional link between soil microbial community structure and N cycling. As a result, we are beginning to understand the microbial community ecology involved in N processes in forest ecosystems, which provide us with a more complete picture of the regulation of the N cycle and its response to environmental change.

4Although some excellent reviews of the ecology of N-cycling microorganisms in soils exist (e.g.,;;;; ), these focus primarily on temperate systems, and the relationship between N processes and microbial community dynamics in tropical forest soils remains poorly understudied. In this review we describe and summarize currently available information regarding the relationship between soil microbial ecology and three relevant N-cycling processes that govern the fate of N in tropical forest soils. Although we also briefly introduce other important microbial N processes, we focus on the N-fixer (diazotrophs), nitrifier and denitrifier communities because these functional groups have been the most studied in tropical soils. We close our review with a discussion of the impact of anthropogenic environmental changes on the microbially mediated mechanisms regulating N cycling in tropical forest ecosystems.

5Tropical soils are generally on older geologic substrates depleted of rock-derived elements such as phosphorus (P) and cations. As a consequence, these soils are typically acidic (pH < 5) and their fertility is often dependent on the cycling of a thin layer of organic matter associated with the large amount of plant litter material. Thus, in contrast to temperate forests, tropical forests on highly weathered soils are assumed to be primarily limited by P and “N-saturated”, leading to an open dynamic N cycle, with relatively high levels of N loss via denitrification and leaching balanced by high levels of N fixation. This assumption is also supported by observations of high plant and soil N/P ratios, high abundance and diversity of legume plants, and the export of bioavailable N at the ecosystem scale (Supplemenatary Table S1;;; ). For example, some fertilization experiments conducted in tropical forests have demonstrated a stronger response of plant biomass to added P than to added N, supporting the paradigm that tropical ecosystems on weathered soils are predominantly P limited. However, there is emerging evidence of limitation and colimitation by other nutrients, such as N and potassium, in tropical forests. For instance, strong colimitation by N and P of vegetation and soil organisms has been observed in an Ecuadorian montane forest. In this forest ecosystem, moderate additions of N (50 kg ha-1 yr-1) and P (10 kg ha-1 yr-1) led to not only an increase in foliar N and P concentrations, but also altered soil microbial biomass, standing fine root biomass, stem growth, and litterfall. Additionally, forest disturbance may lead to N limitation of young secondary forests. Research to date suggests that nutrient limitation in these ecosystems is likely not a question of N vs. P, but instead is the result of complex interactions among multiple nutrient cycles and their linkages with biological processes.

6Soil N transformations in tropical forests are affected by unique soil characteristics (e.g., low pH, rapidly fluctuating redox conditions and large amounts of Fe oxides, plant litter material and available N content) and environmental conditions (e.g., high humidity and low annual fluctuation in temperature), making it difficult to identify the main pathways and mechanisms affecting process-specific transformations (Supplementary Table S1;;;; ). For example, resource stoichiometry influences microbial growth rates and the production of extracellular enzymes that degrade soil organic matter. However, it remains unclear if the pattern of nutrient limitation documented for plants (i.e., strong limitation by P) in tropical forests holds true for soil microbes. For instance, there is evidence that P can limit microbial growth and mineralization in lowland tropical forests found on heavily weathered soils. In contrast, soil microbial biomass and N mineralization in tropical montane forests on younger soils seem to be constrained primarily by N limitation. Furthermore, it has been shown that the response of soil microorganisms to nutrient addition in an Ecuadorian montane rainforest varies with both altitude and duration of nutrient addition. Thus, nutrient availability to soil tropical communities can be affected by multiple factors such as elevation, temperature, rainfall and differences in parent material.

7The complexity of the biological N cycle in tropical forest soils could be depicted by a simplified model (Figure 1) that consists of three main processes: (1) Decomposition of plant litter and dead organisms to soil organic matter, which can be further degraded to dissolved organic N and ammonium; (2) assimilative processes of dissolved organic N, nitrate and by plants and microorganisms for growth and replication; and (3) dissimilative processes including N fixation, nitrification, denitrification, and dissimilatory nitrate reduction to ammonium (DNRA), as well as newly described pathways such as codenitrification and anammox. The pool sizes and rates of production of N available to soil microbes and plants are much greater in many tropical forest soils than in temperate counterparts, due to high rates of N fixation by free-living microbes and rhizobia associated with legumes, which are especially abundant in many tropical ecosystems. This high N availability can exceed the ecosystem N retention capacity and induce high rates of dissimilative processes, with N losses by leaching and emissions of nitric oxide (NO) and N2O.

8The N2O released from soil is of particular concern because it is a potent greenhouse gas. Tropical forest soils are considered the largest natural source of N2O with a source strength of 3.0 Tg N yr-1. The production of N2O in soil has traditionally been assumed to result from the processes of nitrification and denitrification, but growing evidence suggests that other processes may be involved, including nitrifier denitrification, non-denitrifying N2O reduction, DNRA, anaerobic ammonium oxidation (anammox; ), and co-denitrification. Understanding the factors that determine the relative contributions of these different processes (and the microbes that mediate them) to N2O flux has important implications for climate change. However, it is still not clear what underlying processes drive gaseous N production from tropical forest soils. For instance, it has been suggested that nitrification is an important source of N2O in Amazon forest soils. However, some studies conducted in tropical forest ecosystems in Australia, Kenya, and Rwanda suggest that denitrification and nitrifier denitrification could be the main pathways of N2O gas production rather than nitrification. Likewise, anammox bacteria have been detected in a wide range of soil environments, but the role of these organisms in N loss from tropical forest soils has yet to be determined. The DNRA process has also been suggested as a possibly important pathway for production in these soils, because the occurrence of DNRA is generally favored by low and fluctuating redox conditions, such as those in humid tropical forest soils. For instance, found that the rate of DNRA (∼35% of the gross nitrification rate) in a humid tropical forest soil in Puerto Rico was much higher than the rate of N2O production from denitrification.

9There is evidence that tropical forest soils may harbor novel microbial communities, such as unique clades of Acidobacteria and Verrucomicrobia. However, these soils are an especially understudied microbial environment, and very few studies have explored the immense diversity of soil microorganisms in tropical forests or asked if they are phylogenetically or physiologically distinct from those in temperate soils. The majority of these studies have concentrated on the effects of land-use conversion on the structure and function of soil microbial communities (e.g.,;;;; ). Few studies have examined seasonal influences of precipitation, or the effects of plant species diversity or N addition on tropical soil microbial communities.

10Linking microbial community composition, abundance, and activity to rates and controls of biogeochemical transformations is challenging in any ecosystem, but is especially challenging in tropical forest soils. This is due not only to the complexity of tropical forest microbial communities, but also to the high degree of heterogeneity in plant functional diversity and soil chemistry often found in tropical forest ecosystems. For example, rates of many microbially mediated N processes show marked substantial variation across small temporal and spatial scales in tropical forest soils. Tropical plant community composition varies strongly across space in many tropical forest ecosystems, and this can contribute to the high degree of heterogeneity in N cycle processes observed in these systems. For instance, the large amount of plant litter and rhizodeposition (the release of C compounds from living plant roots) in tropical forest soils may enhance microbial activity and stimulate production of microbial exoenzymes that “mine” for more complex soil organic N compounds. The N cycle is also particularly susceptible to changes in soil redox conditions, which are highly variable in tropical forest soils and can cycle between oxic and anoxic on a scale of hours to days. These fluctuating soil redox regimes are a critical determinant of microbial metabolism, and may facilitate the co-occurrence of microbial N transformations with significantly different sensitivities to soil O2 availability, such as nitrification (oxic), denitrification (hypoxic), and DNRA (anoxic). found that the rapidly fluctuating redox conditions in a humid tropical forest soil in Puerto Rico allow anoxic and oxic N processes to co-occur, which are mediated by tolerant and resilient microbial communities adapted to nutrient resources that are spatially and temporally variable.

11A comprehensive understanding of the microbially mediated tropical N cycle is critical because it affects our knowledge of how tropical forests function within the integrated Earth-climate system. For example, quantification and characterization of microbial functional genes involved in N cycling (Table 1 and Supplementary Table S2) can help create informative models of N-cycling rates and their feedback with C dynamics, as well as the impact of N2O emissions from these soils, providing predictions of, and mitigation strategies for, greenhouse gas emissions. We discuss below currently available information regarding microbial functional groups that control the most commonly studied N processes (N fixation, nitrification, and denitrification), with an emphasis on unique aspects of their ecology in tropical forest soils.

12Biological N fixation is the largest natural source of new N to most terrestrial ecosystems. In this process symbiotic and free-living diazotrophic microorganisms reduce atmospheric dinitrogen gas (N2) to reactive and biologically available forms. The nifH gene, encoding the reductase subunit of nitrogenase, the enzyme that catalyzes this reaction, has been widely used as a genetic marker to study the diversity and abundance of diazotrophs. Likewise, a wide variety of PCR primer sets from multiple diazotrophic bacteria have been used to characterize and quantify the nifH gene in soils.

13Relationships between diazotroph communities and N fixation rates have been investigated in many terrestrial ecosystems, including temperate forest soils, wetlands, desert systems, grasslands, and agricultural lands. Despite the fact that tropical forest ecosystems maintain considerable biological N fixation to balance the large potential N losses, this process and the microbial community involved in it remain poorly investigated in these soils. Studies of lowland tropical rainforest in Costa Rica reported that soils were dominated by nifH clones related to the genera Heliobacterium (a member of the Firmicutes), as well as Gluconacetobacter, Methylobacterium, Azospirillum, and Zymomonas (all Alpha-proteobacteria; ). Diazotroph communities in primary and secondary rainforest soils in the western Amazon Basin of Brazil have been reported to be mainly composed of ubiquitous and abundant members of the Alpha and Beta-proteobacteria (e.g., the genera Azospirillum, Azorhizobium, Bradyrhizobium, Methylobacterium, Burkholderia), Firmicutes (e.g., Paenibacillus, Heliobacterium), and Cyanobacteria (e.g., Nostoc, Anabaena; ). Interestingly, nifH sequences associated with methanogenic Archaea (Methanoregula, Methanosphaerula, Methanocella) were also found in these soils. suggested that archaeal biological N fixation could play an important role in the Amazon rainforest.

14Biological N fixation rates in tropical forests (15 to 36 kg N ha-1 yr-1) are similar to or higher than estimates for their temperate counterparts (7 to 27 kg N ha-1 yr-1), which are subjected to strong N limitation. suggest that diazotrophs could be favored in tropical forests because they acquire enough N to maintain higher extracellular phosphatase activity, which is required to overcome P limitation in these ecosystems, and because the temperature in tropical environments is near the optimum for biological N fixation. Furthermore, until recent years it was thought that free-living diazotrophs were the dominant form of N-fixing bacteria in temperate forest soils and symbiotic diazotrophs were the dominant form of N-fixing bacteria in tropical forest soils, where leguminous trees are common. However, recent studies in tropical forests indicate that free-living N-fixing bacteria in litter and soil may be more important than previously thought. Forest canopy communities, especially lichens, mosses, and other epiphytes associated with cyanobacteria, can also provide significant inputs of N to tropical forests. estimated N inputs via biological fixation by free-living microbes from above- and belowground components of two tropical forests in Puerto Rico, and assessed the response to increased N availability using an N fertilization experiment. Their results showed that free-living N fixation in soils was suppressed by N fertilization (50 kg N ha-1 yr-1) and was significantly positively correlated with soil moisture, similar to findings in a tropical rainforest in Panama.

15Tropical ecosystems are characterized by N fixation hotspots —zones where N fixation rates are much higher than average— that may reflect the very small-scale spatial heterogeneity of abiotic factors affecting the dynamics of the diazotroph community. While many tropical soils are generally nutrient-poor, there is a great deal of spatial and temporal heterogeneity in nutrient availability even on highly weathered soils. The distribution and activity of N-fixing organisms in tropical forest soils are mainly regulated by moisture, C/N ratio, and the availability of both P and molybdenum (Mo;;;;; ). Parent material can also drive the abundance of diazotroph communities in tropical soils. found that Inceptisol soils (primary-mineral rich soils) may provide a more favorable environment for diazotroph communities (and also denitrifier communities) compared with their Oxisol (acid soils, rich in Fe and Al secondary minerals) counterparts in two tropical forest types in Puerto Rico. Higher N loss rates due to leaching in Inceptisol subsoils could produce a feedback on surface N-cycle processes, increasing microbial N demand and ultimately stimulating N-fixation. In addition, anaerobic conditions can significantly increase rates of biological N fixation because O2 inhibits the activity of the nitrogenase enzyme, and thus soil moisture content is an important factor in regulating rates of biological N fixation.

16Both P and micronutrients play important roles in regulating N fixation in tropical forests. The high P demand of diazotrophs to provide the large amounts of energy required by this process creates the potential for P limitation of N fixation. In addition, P in many lowland tropical forests is bound up with highly weathered soil minerals and organic matter. Microorganisms are able to liberate this P by exuding phosphatase enzymes and their synthesis requires large amounts of N. Thus, N fixation in tropical forests may confer a competitive advantage to diazotrophs by facilitating the uptake of P. In line with these assertions, some experimental studies have shown that P addition stimulates this process, as well as increases the abundance and diversity of free-living diazotrophs in these ecosystems. Similarly, Mo is a critical component of the most common form of nitrogenase and some studies conducted in the tropical rainforest of Panama have suggested Mo limitation of N fixation. performed a full-factorial litter incubation experiment to explore P and Mo controls over free-living N fixation rates from a Costa Rican tropical rainforest. Results showed that variation in P concentration (and not Mo) was positively correlated with N fixation rates. These authors asserted that more data are clearly needed to draw firm conclusions about general patterns in P vs Mo limitation of N fixation in these soils.

17Nitrification is the stepwise aerobic oxidation of or ammonia (NH3) to nitrite and. This key process plays an important role in regulating N availability and loss in terrestrial ecosystems, because it can cause leaching to groundwater and N2O production directly (via chemical decomposition of hydroxylamine) and indirectly (via denitrification and nitrifier denitrification). Nitrification in soils is divided into autotrophic nitrification and heterotrophic nitrification. The former is mainly carried out by chemoautotrophic ammonium-oxidizing bacteria (AOB, represented by Nitrosomonas, Nitrosospira, and Nitrosococcus species) and archaea (AOA, represented by Nitrososphaera and Nitrosotalea linages from the phylum Thaumarchaeota), and nitrite-oxidizing bacteria (NOB, represented by Nitrobacter, Nitrospina, Nitrococcus, and Nitrospira species). Heterotrophic nitrification is carried out by certain heterotrophic bacteria and fungi with the potential to oxidize both organic and inorganic N compounds. Although it is widely accepted that autotrophic nitrification in soils is the prevalent nitrification process, high nitrification rates in tropical and subtropical acidic soils suggest that heterotrophic nitrification may be important in these soils, because it is less impacted by low pH than autotrophic nitrification. In fact, using the 15N tracing technique, both and found that heterotrophic nitrification was the dominant process producing in subtropical acidic coniferous forest, where autotrophic nitrification rate was very low. Using the 15N tracing method and a selective biomass inhibitor (fungal inhibitor: cycloheximide and bacterial inhibitor: streptomycin), identified that fungal rather than bacterial pathways dominate heterotrophic nitrification in the same forest soils, suggesting that fungi may be the important driver for heterotrophic nitrification in acid forest soils of subtropical China. However, the ecology of heterotrophic nitrifier microorganisms has not yet been investigated in tropical forest soils.

18Nitrifier denitrification is an alternative pathway and involves the oxidation of NH3 to and its subsequent reduction via NO to N2O by AOB, particularly by Nitrosospira taxa. Recent studies have suggested that this process may contribute considerably to N2O fluxes from soils with high N content and low pH and oxygen pressure. These conditions are typical in tropical forest soils, thus we speculate that nitrifier denitrification could play an important role in these ecosystems. However, to our knowledge, no studies directly related to this process have been conducted in tropical forests soils.

19Microbial ammonia oxidation is the first and rate- limiting step of autotrophic nitrification, which may explain why the few studies on microbial nitrification in tropical forest soils have mainly focused on this process. Ammonia-oxidizing microorganisms oxidize NH3 to by using the ammonia monooxygenase enzyme, the α-subunit of which is encoded by the amoA gene, a common genetic marker for nitrification. It was presumed that chemolithotrophic Gamma- and Betaproteobacteria were primarily responsible for this process in soils. However, recent studies have demonstrated that AOA are ubiquitous constituents of terrestrial environments and their discovery has changed the paradigm of aerobic nitrification. The ecology of AOA remains under investigation, and there is not a simple relationship between NH3 oxidation rates and the relative abundances of AOA and AOB.

20In soils, drivers of niche differentiation of AOA vs AOB communities have centered largely on pH, NH3 concentrations and N organic forms. High abundances of archaeal amoA genes have been reported in many acidic soils and multiple studies suggest that some groups of AOA may prefer a pH below 5.5. In strongly acidic soils, AOA abundance increases or remains unchanged with decreasing pH, while AOB abundance decreases. However, in neutral to alkaline soils, correlations of AOA or AOB abundance to pH have not been consistent. Investigations have also indicated a preference of soil AOA communities for low NH3 levels and low organic nutrient conditions.

21Database surveys focused on soils from temperate regions have revealed a strong biogeographical component to the distribution of AOA and AOB phylotypes. However, in tropical soils very little is known about ammonia-oxidizing microorganisms and how environmental factors affect their activity. Many tropical forest soils tend to be wetter, have smaller annual fluctuations in temperature, lower pH, high available N and more reduced conditions than temperate forest soils, and this may impose different environmental conditions (and require different traits) for ammonia-oxidizing organisms. For instance, Nitrosospira cluster 2 has been found in great abundance in acidic soils, but it has been suggested that it may be more common in cold-temperate forest soils than in warmer tropical soils. So far, ammonia oxidizer amoA sequences from clone libraries have been described from only a few wet tropical forest soils from Trinidad, Puerto Rico, and Costa Rica, as well as from two different subtropical forest soils in China. Only three of these studies have successfully amplified bacterial amoA sequences from these soils, with Nitrosospira and Nitrosomonas the primary taxa. The majority of archaeal amoA sequences described in these soils clustered with sequences from Nitrososphaera and Nitrosotalea.

22Similar to many acidic temperate forest soils, AOA populations have been inferred to be the primary group driving nitrification in tropical forest soils. Furthermore, and were unable to amplify bacterial amoA genes from acidic tropical forest soils (Supplementary Table S2). At least two other studies examining acidic forest soil have found a similar pattern of absence of bacterial amoA genes. Thus, one possible explanation for the dominance of AOA in these soils is the relatively low soil pH. For example, observed that the ratio of AOA to AOB amoA gene abundance increased with decreasing soil pH in subtropical soils in China. Significant relationship between AOA abundance and nitrification potential was also found, indicating that nitrification was mainly driven by AOA in these acidic soils. However, found that AOA abundance predominated over AOB in tropical soils that spanned pHs of 4 to 8 in Trinidad (Supplementary Table S2), and the community abundance and structure of AOA was affected primarily by soil N characteristics, but not by soil pH. According to these authors, the effect of pH on nitrification appeared to mainly reflect impacts on AOA or AOB activity, rather than selection for AOA or AOB phylotypes differing in nitrifying capacity. In addition, found higher net nitrification rate and abundance of AOA and AOB for Cambisols than for Ferralsols from the same parent material in a subtropical acidic forest in China (Supplementary Table S2). These authors also reported higher -N immobilization for Ferralsols than for Cambisols, which imply that the lower nitrification rate and amoA abundance in these Ferralsols may due to high amounts of -N immobilization stimulated by high Fe oxide concentrations. A potential mechanism of abiotic immobilization of NO3 has been postulated, suggesting that Fe plays a key role in -N immobilization by promoting organic N formation from inorganic N. Thus, amoA genes abundance may be more affected by soil nutrients and Fe oxide contents rather than a low pH in tropical forest soils.

23In contrast to temperate forest soils, tropical soils routinely experience fluctuating O2 availability over short time scales. This could represent a unique selective habitat for ammonia oxidizers adapted to this O2 regime. Laboratory measurements in Puerto Rican forest soils showed elevated pools and high gross nitrification rates immediately following periods of anaerobiosis, indicating that substantial oxidation occurs despite low pH and limited O2 availability. The survival mechanisms of these nitrifiers during periods of prolonged anaerobiosis are not well known. It has been shown that under oxygen-limiting conditions, ammonia oxidizers can use as a terminal electron acceptor, or exploit the oxic-anoxic spatial segregation. Another explanation is that is constantly regenerated via DNRA due to the redox fluctuation conditions in tropical soils, and with a subsequent influx of O2, this becomes reavailable to ammonia oxidizer communities. On the other hand, AOA populations appear to be insensitive to O2 availability. Tropical soil AOA not only tolerate extended periods of low O2, but can react faster than AOB in alternating aerobic/anaerobic conditions. It has also been proposed that the pathway of oxidation in AOA may be distinct from the AOB pathway, theoretically requiring only 0.5 O2 per oxidized. This hypothesized lower O2 demand could explain why AOA populations seem to persist more readily under low-oxygen conditions.

24A lack of correspondence between nitrification rates and nitrifier community abundance in tropical forest soils has been reported by some investigators, and the reasons for this disconnection are unknown. It is possible that unique ammonia-oxidizers exist in these ecosystems, which can evade detection by the PCR primers developed from currently available sequence data. It is tempting to speculate that the particular conditions of these soils may select for specific lineages of ammonia oxidizers, although much more molecular microbial diversity data from these soils and additional characterization of soil isolates are needed to test this hypothesis.

25Denitrification is a microbial anaerobic respiration pathway through which or are sequentially reduced to N2 via the intermediates NO and N2O. It is the major biological process in soils that returns fixed N to the atmosphere and closes the N cycle. It is also the primary pathway of NO and N2O emissions from soil. Denitrification consists of four reactions catalyzed by the metallo-enzymes nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase. Nitrite reductase is the key enzyme of denitrification, since it catalyzes the reduction of to gas products, which is the reaction that limits the rate of the denitrification process. Several studies have used genetic markers for denitrification, including narG and napA (markers for nitrate reductase), nirK and nirS (markers for nitrite reductase), norB (a marker for nitric oxide reductase) and nosZ (a marker for nitrous oxide reductase), to study the diversity of denitrifying microorganisms in various environments. Denitrifier organisms can have different combinations of genes involved in the denitrification pathway. Likewise, while some microorganisms harbor all denitrification enzymes and can potentially perform the complete pathway, others lack the nitrous oxide reductase gene and produce only N2O as the denitrification end product. Furthermore, recent studies have identified non-denitrifying N2O reducers with atypical nosZ, which are potential contributors to N2O reduction in soils. Thus, understanding the ecology of this functional guild is potentially important for mitigating N2O emissions from soils. This newly identified clade of N2O reducers is diverse and widespread in terrestrial environments, but the ecology of this group in tropical forest soils is still unknown.

26Denitrification is distributed among a taxonomically diverse group of microorganisms, which have acquired the ability to denitrify via different evolutionary mechanisms. This process in soil is mainly performed by facultative aerobic heterotrophic bacteria, such as some species of Pseudomonas, Bacillus and Paraccocus, and autotrophic bacteria, such as Thiobacillus denitrificans. Denitrification is also found among a few archaea and fungi, including Ascomycota (e.g., Fusarium oxysporum, Fusarium solani, Cylindrocarpon tonkinense and Gibberella fujiuroii) and Basidiomycota (e.g., Trichosporon cutaneum). Fungi generally lack N2O reductase, suggesting that these organisms are responsible for a large portion of N2O emission from soil. Despite the frequent observation that tropical microbial communities in soil are often dominated by fungi, their contributions to N2O emissions from tropical forest soils have not yet been addressed. The polyphyletic distribution of denitrifying genes can result in their co-occurrence with N-fixation and ammonia-oxidation genes in many microbes. Many diazotrophic bacteria, such as some species of Azospirillum and Bradyrhizobium, can denitrify. Similarly, AOB belonging to either the genus Nitrosospira or Nitrosomonas have been shown to be capable of denitrification.

27Understanding the link between ecosystem scale denitri fication rates and the ecology of the microorganisms that mediate this pathway has been identified as a critical research goal for the mitigation of climate change. For this reason, numerous studies have been conducted on the microbially mediated denitrification process in temperate soils, especially its role in N losses and its sensitivity to various environmental factors. Despite the growing evidence that denitrification mechanisms and denitrifier communities in tropical forest soils may be very different from those of temperate soils (due to their unique soil characteristics and environmental conditions;; ), few studies exist that document the relationship between denitrifier abundance and total N gas production in tropical and subtropical soils (;;;; Table 1; Supplementary Table S2). Furthermore, contradictory results have been published about the importance of denitrification as a significant route of N loss in tropical ecosystems. Some studies have shown that tropical forest soils are characterized by generally lower denitrification capacity than temperate forest soils, with greater variability both temporally and spatially due to heterogeneity in edaphic conditions. However, tropical soils are considered to be the largest natural sources of NO and N2O due to the combination of climatic factors, soil properties, and rapid rates of N cycling. The conceptual “hole-in-the-pipe” model gives a simplified view of the main controls on N2O and NO fluxes. In this model, the rate of nitrification and denitrification is conceptualized as a pipe, while the relative ratio of NO and N2O to total gas produced is conceptualized as a hole. According to, N flowing “through the pipe” is significantly less in subtropical forest soils than in temperate forest soils. However, the “holes in the pipe” are much bigger in subtropical forest soils than in temperate forest soils, resulting in a larger amount of NO and N2O gases “leaking out of the pipe”. High redox potential (resulting from enrichment of iron and aluminum oxides), low pH and high N availability are the key factors affecting the amount of N flowing through the pipe and the size of the holes in the pipe. These factors may suppress complete denitrification in humid tropical soils and enhance NO and N2O ratios during denitrification.

28The quantification of denitrification genes to elucidate the relative importance of abiotic environmental factors versus denitrifier distribution has been recently achieved in tropical forest ecosystems (;;;;; Table 1; Supplementary Table S2). These studies indicate that many soil properties can affect denitrifier community attributes and denitrification rates in tropical soils, including parent material, pH, redox potential, O2 partial pressure, available organic C and N, and concentration. For example, the abundances of denitrification genes (nirS and nosZ) were positively correlated with soil C, N, and P concentrations and were more abundant in the Inceptisol compared with the Oxisol soil type in humid tropical forests in Puerto Rico, suggesting more dynamic N transformation processes in the sandier soil sitting on a younger, more rapidly weathering parent material. found that the abundance of both norB and nosZ gene copies were positively correlated with soil pH, and NO and N2O fluxes from acidic forest soils (pH 4.4-4.7) were lower than in farmland soils with higher pH (5.0-5.3) in a subtropical region of China. On the other hand, showed that redox potential, rather than soil pH or organic C, was the key soil variable influencing denitrification capacity and the abundance of denitrifiers in subtropical forest soils from China. Plant species composition has also been suggested as an important factor in determining the distribution and abundance of denitrifiers in soil. So far, only one study has addressed the influence of plant species composition on the abundance of soil N functional genes in tropical soils. These authors found that inter-planting teak with fluegge increased N stocks and the relative abundance of denitrification genes compared to teak mono-plantations in the Salomon Islands, suggesting that mixed species plantations may potentially alleviate N losses and favor N retention.

29found that pristine rainforest soils in Amazonia showed higher N content and abundances of denitrifier genes (nirK and nosZ) than pasture and crop fields, which correlated with N2O emissions. However, found no correlation between denitrifier abundance and N2O fluxes in a tropical forest soil in Australia, suggesting that soil C and N substrate availability and soil environmental factors (pH and moisture) rather than denitrification gene abundance control N2O fluxes in this ecosystem. Studies in other soil environments have also reported that changes in denitrification rate were not associated with changes in denitrifier community structure. This may be attributed to the high functional redundancy commonly found in phylogentically diverse groups such as denitrifying bacteria. A further understanding of this topic requires research on the expression of denitrification genes (e.g., via RNA-based approaches that target physiologically active populations in denitrification) and its relation to denitrification rates, as well as to separate N2O produced through denitrification from nitrifier denitrification.

30It is well known that O2, organic C and availability, low pH, or sudden onset of anoxia can also influence denitrification rates and negatively affect the reduction of N2O to N2. However, the mechanisms that increase gene transcription and enhance N2O reductase activity have not been researched in detail in tropical forest soils. For example, soil pH is one of the main factors controlling broad-scale microbial activity and the stoichiometry of denitrification. This process in acid soils, such as typical tropical forest soils, seems to be particularly disposed to high N2O/N2 ratios, apparently due to impaired functioning of N2O reductase at low pH. However, contradictory results have been published on the relationship between soil pH and denitrification rates in tropical and subtropical acid soils in China. showed that denitrification capacity was not correlated with soil pH in humid subtropical soils, but with organic carbon content. Furthermore, no correlation between soil pH and denitrification rate was found in six forest soil types in different climatic zones (temperate, tropical and subtropical forests; ). On the other hand, reported that low soil pH (<4.7) decreased denitrification rates and NO and N2O emissions in subtropical forest soils from China.

31Soil moisture is a major driver of N2O emissions as it regulates the oxygen availability to soil microbes. N2O emissions have their optimum in the range of 70–80% water-filled pore space depending on soil type. Since denitrification occurs as soil O2 becomes limiting, denitrifiers are likely to be more widespread and resilient in tropical soils due to their temporally and spatially variable redox environment. These taxonomically and physiologically diverse facultative microorganisms preferentially respire O2, but can reduce and other partially oxidized N-forms when O2 is limiting. performed a fluctuating redox incubation experiment using tropical forest soils from Puerto Rico and found that denitrification tended to be highest in soils that had been anoxic for several weeks. These results indicated that denitrifiers might dominate in situations where O2 is consistently limiting, whereas microbes that mediate DNRA may be more competitive for in a fluctuating redox environment. On the other hand, frequent oxic/anoxic transitions may also inhibit the expression of nitrous oxide reductase, since its activity is more sensitive to O2 compared with other denitrifying enzymes.

32Alteration of the N cycle by human activities affects climate, food and energy security, human health, and ecosystem services. Anthropogenic factors also have a great potential to alter N processes in tropical forests and significant changes in N dynamics in these forests is expected, including rising export to ground and surface waters, threatening drinking water quality and increasing eutrophication risks. Furthermore, in the coming decades, the tropics will experience unprecedented increases in temperature and significant alterations in precipitation regime, which also have the potential to affect the N cycle. For example, soil warming may increase microbial metabolism and enzyme activity, resulting in greater organic matter decomposition and gross N mineralization. Warmer temperatures could also stimulate additional N losses via increased denitrification rates. However, so far very little is known regarding the response of microbial communities involved in N processes to different components of global environmental change in the tropics. It is beyond the scope of this paper to review the myriad microbial responses to environmental change; rather, we focus our review on the limited currently available research regarding the response of microbial N cycling to anthropogenic factors in tropical forest soils, such as chronic-elevated N deposition, forest perturbation, and land-use change.

33Atmospheric deposition of reactive N compounds has increased threefold during the last century due to fossil fuel combustion, artificial fertilizer application and leguminous crops. Currently, N deposition adds more than 200 Tg yr-1 of N to terrestrial and ocean ecosystems, and is predicted to increase between 50 and 100% by 2030 compared with 2000. Furthermore, reactive N deposition is dramatically increasing in the tropics because of economic development and increasing biomass burning, resulting in many unexpectedly negative effects. In fact, elevated N deposition not only occurs in areas with high population densities but also in areas far from the source of N. For example, southern Ecuadorian montane forests receive elevated atmospheric N and P deposition that is largely attributed to biomass burning in the Amazon basin. In addition, recent climate models that incorporate N deposition as a part of the CN-coupled models predict a lower net C uptake (37–74%) than values projected by models using C cycle components alone, demonstrating the importance of the N cycle in future climate change. Thus, a priority in terrestrial ecosystem research is to determine the fate of N, its interactions with soil microbial communities and its coupling with other biogeochemical cycles, such as the C cycle.

34The responses of soil N transformations to N addition depend on forest N status, soil characteristics, and climatic factors. Anthropogenic N deposition has been shown to greatly affect N cycling in temperate and boreal forests, where low availability of N limits primary productivity. These responses have been well studied and summarized in the conceptual model known as the “N saturation hypothesis”. The concept of “N saturation” refers to a state in which N supply exceeds biological demand, resulting in the loss of new N from the ecosystem by biological or physical mechanisms. In this model, atmospheric chronic N deposition in temperate and boreal forests increases soil acidification, stimulates N loss through extensive leaching and gaseous N emissions, and produces a switch from N to P limitation and a decline in plant diversity. In contrast to temperate forests, tropical forests are generally considered N-rich relative to other nutrients, and thus N deposition is less likely to increase primary productivity of tropical ecosystems, but may alter other aspects of the N cycle. It has been also hypothesized that chronic N deposition will acidify tropical forest soils, further exacerbate P deficiency in highly weathered soils, reduce tree growth and C storage, and negatively affect biodiversity. Recent evidence suggests that these changes are currently occurring in tropical forests.

35Compared with other ecosystem types, tropical forests emitted more N2O under N enrichment (on average +739%; ), which suggests strong feedbacks of soil N2O emissions on increasing atmospheric N deposition in the tropics. However, research on the effect of N deposition in tropical and subtropical forest ecosystems is much more recent, and the temporal scale of such studies much shorter than those in temperate forests. For example, the effect of N deposition on N-saturated tropical forest ecosystems in southern China has been recently studied. Forests in this region have acidified soils, exhibit chronic atmospheric N deposition of 16-38 kg⋅ha-1⋅yr-1 and high throughfall N input that vary greatly from 11.7 to 65.1 kg N⋅ha-1⋅yr-1. It had been shown that N2O emission significantly increases, but soil pH, primary production, litter decomposition and N mineralization significantly decreases in high N addition (100-150 kg N⋅ha-1⋅yr-1) plots. These results are consistent with progressive P limitation, which results in higher litter N content and soil N availability. Likewise, a two-year N addition experiment (50-150 kg N⋅ha-1⋅yr-1) was performed in a humid subtropical bamboo forest in China, which exhibits one of the highest chronic atmospheric N deposition rates in the world (113.8 kg N⋅ha-1⋅yr-1 in 2009; ). In this ecosystem, N deposition accelerated nitrification and denitrification rates, increased hydrolytic enzyme activities, soil N availability and microbial biomass, and decreased soil P availability. Results suggest that this bamboo forest ecosystem is moving toward P limitation under elevated N deposition, and may increase N2O emission and leaching.

36The same pattern of increased N-oxide emissions and leaching induced by N deposition has been found for montane tropical forests. In Hawaiian montane forests, 11-13 years of high N addition (100 kg N⋅ha-1⋅yr-1) to an N-limited forest on an Andosol soil and 5-7 years of high N addition (100 kg N⋅ha-1⋅yr-1) to a P-limited forest on a Ferralsol soil increased gross rates of mineral N production but decreased microbial N retention, leading to higher soil N-oxide emissions and leaching. Similarly, 1-4 years of high N addition (125 kg N⋅ha-1⋅yr-1) in an N-limited Panamanian montane forest on an Andosol soil increased gross rates of soil N mineralization and nitrification, leading to increases in soil N-oxide emissions and leaching. also showed that this wet montane forest with only 4 years of N addition had comparable N-oxide emissions to an N-rich moist lowland forest in Panama with more than a decade of N addition.

37Elevated N deposition in tropical forests may also significantly change soil microbial community composition and activity. There are two primary hypotheses that seek to explain the influence of N addition on microbial community dynamics. The N-mining hypothesis proposes that under N enrichment, microbes reduce decomposition of recalcitrant C in response to their lowered N requirements, resulting in a shift toward labile C decomposition and reduced overall microbial activity. The copiotrophic hypothesis suggests that N additions decreases the relative abundance of oligotrophic taxa (k-strategists, i.e., slow growing microbes that have high substrate affinity and can catabolize more recalcitrant C pools), which would be out-competed by more copiotrophic taxa (r-strategists, i.e., fast growing microbes that have high nutritional requirements and preferentially consume labile soil organic C pools;,; ).

38Knowledge about the effects of N deposition on particular microbially mediated N processes in tropical forests is still limited and controversial (Table 1). reported that N fertilization (50 kg N ha-1 yr-1, which is approximately twice the average projected rate for Central America for the year 2050; ) suppressed soil biological N fixation in two tropical forests in Puerto Rico, similar to findings in a rain tropical forest in Panama. investigated nitrifying communities of acidified subtropical forests soils in southern China and found that archaea rather than bacteria control oxidation after long-term N deposition (32-34 kg N⋅ha-1⋅yr-1). However, it has been reported that rates of heterotrophic nitrification were significantly higher than those of autotrophic nitrification in these forest soils. found that the high relative abundance of both amoA and denitrification genes, especially narG, in a tropical rainforest soil in China was due to the large amount of soil available N (from 141.4 to 224.5 mg kg-1), caused by the high rate of N deposition (over 25 kg N⋅ha-1⋅yr-1) that affects this ecosystem. In sum, there is evidence indicating that N deposition in tropical forest soils may inhibit biological N fixation, alter N mineralization and nitrification, and induce more N2O emissions and leaching, particularly during the wet season. However, the consequences of these changes at the ecosystem scale are not well understood.

39We summarize all available evidence in a conceptual model of how increasing N deposition may affect microbially mediated N processes in N-rich tropical forests soils (Figure 2). We hypothesize that chronic-elevated N deposition may lead to a decrease of biological N fixation, N mineralization and DNRA, as well as an increase of heterotrophic nitrification and denitrification, which will lead to high N losses via N leaching and gaseous N production. Furthermore, we also emphasize that these processes are affected by multiple factors such as the initial soil N status, soil type, soil moisture and temperature, elevation (montane or lowland), and landscape position, which may alter the responses of these processes to N deposition.

40Land use change is considered the most important factor driving biodiversity losses in tropical areas. In recent decades, tropical forests have undergone rapid conversion to agriculture, with 80 million hectares of forest cleared in the 1980s and 1990s. Logging and forest conversion result in a series of ecosystem alterations that include an increase in belowground productivity, changes in soil chemistry, and a more open N cycle. Following deforestation, not only plant biomass but also the soil stocks of total C and N can become mobilized and be emitted in the form of greenhouse gases. Among these emissions, N2O has a global warming potential for 100 years 265-298 times greater than that of the equivalent amount of CO2 and promotes ozone destruction in the stratosphere. Thus, understanding the controlling factors of N2O emission is crucial for quantifying the effects of land-use change on the N cycle and for managing and mitigating the environmental consequences associated with N pollution.

41It has been shown that land use change in the tropics also alters soil bacterial, fungal, and archaeal community composition. For example, a recent study showed that the conversion of Amazonian forest to pasture impacts microorganisms, resulting in biotic homogenization of communities. Various studies have suggested that deforestation of tropical forests for agriculture and agroforestry decreases microbial biomass and alters the soil microbial composition. However, the impact of land use changes on soil N processes and the microorganisms responsible for these processes has been limited to only a few studies (e.g.,;;; Table 1; Supplementary Table S2).

42So far, only one study has integrated measures of greenhouse gas emissions and the abundance of multiple functional and phylogenetic genetic markers (nifH, amoA, nirK, nirS, norB, nosZ, mcrA, pmoA, and 16S/18S rRNA) with environmental parameters to predict the response of biogeochemical processes to land use change in Amazonian soils. Results showed that land use change from forest to agriculture reduced the abundance of different functional microbial groups related to C and N cycles in the wet season, but its effects were less significant after the dry winter fallow. studied the influence of land use change from forest to pasture on functional gene diversity, composition and abundance in Amazon rainforest soil microbial communities. They found that genes linked to C and N cycling were the most altered by deforestation. also observed that soil AOB diversity and composition changed significantly with land use in the tropical lowland of Costa Rica, and these changes were significantly associated with shifts in the potential rates of nitrification. The work of is one of the first studies using the marker gene nifH to assess the response of free-living diazotrophs to deforestation in the Amazon rainforest. They reported a shift in the diazotrophic community composition and an increase in nifH gene abundance following forest-to-pasture conversion. They suggested that the response of diazotrophs to land use change is a consequence of changes in plant communities, particularly the higher N demand of pasture for supporting aboveground plant growth. also found that the abundance of denitrifying genes (narG, nirK, norB, and nosZ) was significantly affected by land-use change in subtropical soils of China.

43Most studies of the consequences of N cycle alteration induced by anthropogenic environmental changes have been performed in temperate forests where biological processes are limited by N supply. However, the response of N processes to environmental change in tropical forest soils is largely unknown. In order to better understand, predict and mitigate global environmental changes, there is an urgent need to clearly understand the microbial control of greenhouse gas emissions and its interactions with environmental factors. Furthermore, a deeper understanding of microbially mediated N processes in tropical forest soils is crucial as N cycling in these soils impacts food production for a large part of the world, as well as the dynamics of global climate change.

44This review reveals large knowledge gaps regarding micro bially mediated N processes in tropical forest soils, gaps that represent critical directions for future research. We propose several topics of research that should be prioritized (Box 1). For example, to more accurately predict the response of N cycling to environmental change in tropical forest ecosystems, additional data regarding how N cycling is regulated is needed from a much broader range of tropical forests than currently studied, as well as improvements in our ability to quantify in situ the rates of the primary N processes in these soils. Likewise, it is important to carry out studies in tropical forest soils of recently discovered processes, such as anammox and codenitrification. In addition, research is required to link these processes with soil microbial community characteristics and the functional roles of microbial extracellular enzymes, in order to provide mechanistic insights into microbial regulation of the N cycle in tropical forest ecosystems.

45• The contributions of different microbial subpopulations to the N cycle.

46• The ecology of recently discovered processes, such as anammox and codenitrification.

47• Modeling microbial regulation of the N cycle.

48• Network analyses to explore links between environmental factors and microbial control of the N cycle.

49• Links between specific microbial metabolic processes and the C and N cycles.

50• The interactive effects of C, N, and P on the soil microbial community.

51• The influence of precipitation and soil warming on the community structure of N-cycling microorganisms.

52• The influence of soil spatial heterogeneity on microbial community functions and N cycling regulation.

53• The impact of disturbance on microbial community structure and function and its consequences for the N processes at global scales.

54• Microbial community responses to multiple experimental climate change drivers.

55• Mechanisms regulating the activity and interaction between primary producers and microorganisms controlling N cycling (plant-microbial feedbacks for the regulation of N).

56SP and BB designed and conceived the study. SP produced the first draft of the manuscript, and both authors edited the manuscript.

57The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.