The Main Influencing Factors And Mechanism Of Soil Heterotrophic Nitrification

Heterotrophic nitrification and autotrophic nitrification are two important pathways for soil NO₃^-production.However,there are still uncertainties about heterotrophic nitrification due to the various heterotrophs and nitrifying substrates.Recently,heterotrophic nitrification has been recognized as an important process for NO₃^-and N₂O production in various ecosystems.From the currently published reports in cropland,forest and grassland ecosystems,a high proportion of heterotrophic vs.total nitrification is often observed in soils that are characterized by low pH values,typically between 4.0 and 6.5,and C:N ratios>10.The correlation would provide theoretical basis for predicting the occurrence of heterotrophic nitrification and establishing the evaluating model of N₂O,if the relationship were confirmed.Therefore,the study was aimed to explore the effects of soil pH and C:N ratio on heterotrophic nitrification and the N₂O production via this pathway.Typical subtropical?SF?and temperate?TF?forest and cropland soils?SC and TC?were selected.Then ¹⁵N labeling technique which is used to quantify gross N transformation rates and N₂O production pathways,coupling with molecular quantification that is applied to illustrate the microbial information?abundance and species?was conducted to illustrate the occurrence and mechanism of soil heterotrophic nitrification.It is generally believed that heterotrophic nitrification occurs ubiquitously in acidic soils and is important for NO₃^-production.However,whether low soil pH stimulates heterotrophic nitrification is unknown and the potential microbial driver behind it is unclear.To illustrate the regulation of soil pH on heterotrophic nitrification,a pH gradient?3.5,4.5,5.5,6.5,7.5?was used to manipulate forest?SF?and cropland?SC?soils in subtropical China.During 30 days of pH manipulation,soil microbial information?gene abundance of fungi,bacteria,ammonia-oxidizing archaea?AOA?-amo A,and ammonia-oxidizing bacteria?AOB?-amo A,gene composition of fungi and bacteria?was collected.After 30 days of pH regulation,1%C₂H₂ was used to inhibit autotrophic nitrification and distinguish heterotrophic nitrification from soil nitrifying activity in ¹⁵N-labelling experiments.The results showed that the gross heterotrophic nitrification rates increased from<0.3 mg N kg^-1 day^-1 in pH 7.5treatments to>1.0 mg N kg^-1 day^-1 in the pH3.5 treatments,and the contribution of heterotrophic nitrification to the total NO₃^-production was enhanced to more than60%in the low pH treatments in both SF and SC.With soil acidification,more organic than inorganic nitrogen substrate was used in heterotrophic nitrification.Fungi showed a significantly positive correlation with the gross heterotrophic nitrification rate?P<0.01?and with the contribution of heterotrophic NO₃^-production to total NO₃^-production?P<0.01?,suggesting that fungi are the dominant heterotrophic nitrifiers in acidic soils.In addition,Phialocephala,Chloridium,and Tararomyces may have the potential for heterotrophic nitrification in our studied acidic soils.The present study illustrated the stimulatory effect of low soil pH on fungal abundance and composition,and heterotrophic nitrification.Apart from low soil pH,a high carbon to nitrogen?C:N?ratio is generally considered an important factor inducing heterotrophic nitrification in acidic soils.However,few studies have investigated the regulation of C:N ratio on heterotrophic nitrification.In the present study,a C:N ratio gradient?23,19,15,10?was manipulated in SF and SC under 24 hours and 30 days incubation,and ¹⁵N-labeling techniques combined with C₂H₂ inhibition were used to distinguish between heterotrophic and autotrophic nitrification.Gene copy abundance and soil fungi and bacteria composition were also determined under 30 days C:N ratio regulation.The results showed that the gross heterotrophic nitrification rate was highest in CN23treatment in SC(average 0.80 mg N kg^-1 day^-1),significantly decreasing with declining C:N ratio?P<0.01?.While in SF,the gross heterotrophic nitrification rate was significantly higher in CN15(2.80 mg N kg^-1 day^-1)and CN10(1.38 mg N kg^-1day^-1)treatment than in CN23(0.17 mg N kg^-1 day^-1)and CN19(0.42 mg N kg^-1 day^-1)treatment?P<0.01?.The results indicated that N input in C-abundant soils?e.g.SF in this investigation?and C input in N-abundant soil?e.g.SC in this investigation?,instead of high C:N ratios,may stimulate soil heterotrophic nitrification.The stimulatory effect of acetic acid on heterotrophic nitrification in SC rather than SF further confirmed our conclusion.Soil fungal populations were significantly and positively correlated with gross heterotrophic nitrification rates and the ratio of heterotrophic nitrification rate to total nitrification rate,in addition to the contribution¹⁵N-Glycine treatments,indicating soil fungi could play the key role in driving heterotrophic nitrification.In addition,our results suggested that Mortierella and Trichoderma were the potential fungal species driving heterotrophic nitrification in acidic forest soils,while Exophiala seemed to be the fungal species driving heterotrophic nitrification in acidic agricultural soils according to their high abundance in C-abundant treatments.Consequently,initial soil C and N status should be taken into account when predicting the responses of heterotrophic nitrification to C or N soil inputs.Since heterotrophic nitrifiers could oxidize both N and C substrate.Therefore,the availability of soil organic C is also suggested an important factor regulating heterotrophic nitrification.It is assumed that heterotrophic nitrification would be enhanced when soil organic carbon?SOC?was scarce or recalcitrant.To test this hypothesis,N transformations were quantified in five cultivated soils?SC?and six forest soils?SF?in subtropical region,ten cultivated soils?TC?and seven forest soils?TF?in temperate region in China.Subsequently analysed with respect to the soil physicochemical properties?pH,C:N ratio,SOC,TN?,the chemical compositions of SOC?recalcitrant and labile organic C?and microbial communities?abundance of fungi,bacteria,amo A gene of AOB and AOA?.The results showed that a significantly higher heterotrophic nitrification rate was observed in TC(0.52 mg N kg^-1 day^-1)compared to the other soils.The total content of soil C and C:N ratio could not explain the occurrence of heterotrophic nitrification,but recalcitrant C fractions of carbonyl C and aromatic C,and the ratio of recalcitrant C to labile C?R:L ratio?was significantly and positively correlated with heterotrophic nitrification rates?P<0.01?.This was further confirmed by structural equation modeling?SEM?that the R:L ratio was the most important indicator,suggesting that chemical composition of SOC is likely to be key to explain and predict heterotrophic nitrification rates across a large range of soils including soils high in black carbon such as chernozems,old grassland soils and/or anthropogenic soils such as anthrosols?Terra preta?.In addition,heterotrophic nitrification rates were significantly and positively correlated with fungal gene copy numbers?P<0.05?,providing a strong indication that fungi might be the predominant organisms carrying out heterotrophic nitrification.Subtropical acidic forest soil is an important source of global nitrous oxide?N₂O?.However,the rates of autotrophic nitrification and denitrification in such soil are less than in temperate forest soils.We hypothesized that this difference is related to different N₂O production pathways.We carried out paired ¹⁵N-labelling experiments under aerobic conditions?60%water-holding capacity?in the laboratory to compare N₂O production pathways between SF and TF to determine the differences in N₂O production based on the controlling factors.Our results showed that the contributions soils?21–30%?.Contributions of denitrification to total N₂O production?Cd?in SF?34%?were significantly less than those in TF?54%?.However,rates of N₂O emission from denitrification?N₂O_d?were similar between SF and TF,indicating that denitrification was probably not the predominant process causing the difference between the rates of N₂O production in these soils.The average contribution of heterotrophic nitrification to total N₂O production?Ch?was significantly larger in SF?45%?than in TF?15%?.Soil pH and the C:N ratio were identified as key factors,with Ch negatively correlated with soil pH?r=-0.60,P<0.05?and positively correlated with soil C:N ratio?r=0.78,P<0.01?.The rate of N₂O production by heterotrophic nitrification?N₂O_h?(average 3.0?g N kg^-1 day^-1)was also significantly larger in SF than in TF(average 0.7?g N kg^-1 day^-1).Therefore,based on this study,the heterotrophic N₂O pathway seems responsible for the larger N₂O production in SF than in TF.This study is among the first to elucidate in detail contributions to the processes of N₂O production and to account for its large rates of production in subtropical acidic forest soil in China,which has implications for the prediction of N₂O production in forest soil using ecosystem modelling.To test the stimulatory effect of low pH on N₂O production via heterotrophic nitrification,a ¹⁵N tracing study was carried out on SF and SC in 30 days pH gradient treatments to evaluate the effect of soil pH on N₂O production pathways.Gene abundances of autotrophic nitrification?AOA-amo A,AOB-amo A?,heterotrophic nitrification?bacterial 16S r RNA,fungal ITS r DNA?and denitrification?nir K,nir S,nos Z?-related N₂O production were determined together with structural equation modelling?SEM?to identify relationship between soil pH,functional genes and N₂O production pathways.The results showed that the heterotrophic nitrification?N₂O_h?-derived N₂O in SF was significantly higher in pH3.5(average 4.36?g N kg^-1day^-1)and pH 4.5(average 4.61?g N kg^-1 day^-1)treatment compared with other pH treatments?P<0.01?.The N₂O_h in SC also significantly decreased from 5.74 in pH3.5 denitrification-derived N₂O?N₂O_d?were also negatively correlated with pH,while autotrophic nitrification-derived N₂O?N₂O_a?increased with pH in both SF and SC.For N₂O_d,nos Z played a significantly negative but prevailing role in comparison with nir K and nir S.And N₂O_d was more directly affected by nos Z rather than pH.In comparison with AOA,AOB respond to pH variation more sensitively and had a pronounced effect on N₂O_a when pH was the main controller.However,the role of soil fungi and bacteria on N₂O_h could not be determined in this study.To test the effect of high soil C:N ratio on N₂O production via heterotrophic nitrification.A SF and SC was selected,associated with the soil nitrification?AOA-amo A,AOB-amo A,fungal ITS r DNA,bacterial 16S r RNA?and denitrification-related?nir K,nir S,nos Z?genes in 30 days C:N ratio manipulation.And a 24 hours C:N ratio regulation experiment,including the addition of acetic addition,was also conducted to testify the results in 30 days regulation.Results showed that the heterotrophic nitrification-derived N₂O?N₂O_h?increased from 2.37 in the CN23 treatment to 4.02?g N kg^-1 day^-1in the CN10 treatment in SF,while N₂O_hdecreased from 4.14 in the CN23 treatment to 3.42?g N kg^-1 day^-1 in the CN10treatment in SC.The results in the 24-hour incubation were consistent with 30-day incubation,and the addition of acetic acid significantly increased N₂O_h compared with CN10 treatment in SC?P<0.05?,while not in SF(0.87 in CN19ac vs.0.87?g N kg^-1day^-1 in CN19 treatment).In addition,the responsible heterotrophs?i.e.,fungi and bacteria?were negatively and positively correlated with C:N ratio in SF and SC,respectively.Consequently,C:N ratio is not a strong predictor for soil N₂O production,but the initial C or N status and composition of functional genes may provide important information in acidic soils.These results in this investigation illustrate heterotrophic nitrification is responsible for the significantly higher N₂O production in SF than TF;low pH stimulates soil gross heterotrophic nitrification rates and enhances N₂O production via heterotrophic nitrification;C:N ratio is not sufficient to explain soil heterotrophic nitrification and its N₂O production.In contrast,soil C or N status and the chemical composition of SOC?e.g.labile vs.recalcitrant SOC?is important for predicting soil heterotrophic nitrification and its production.These results are helpful to understand the favorable conditions for soil heterotrophic nitrification,as well as their microbial mechanisms.The process-based data provides information for soils in which certain N₂O production pathways are prevailing,and is essential for estimating global N₂O emissions and developing mitigation techniques.