The Quest For The Source Of NH₃ During Nitrification In Acidic Soils

Nitrification is the only process that links the reduced N and the oxidized N pools, which plays a key role in maintaining global nitrogen balance. Nitrification, a typically biological process, is also highly sensitive to p H. Previously, nitrification in acidic soil is considered to be an evidence of the heterotrophic nitrification, but later studies have shown that autotrophic nitrification is the main process in the acidic soil. The ammonia monooxygenase（AMO） can only catalyze the non-ionic ammonia（NH3）, not the ionic ammonium（NH₄⁺） in autotrophic nitrification. However,the concentration of NH3 is very low in acidic soil, so the sources of the substrate NH3 in acidic soil are widely concerned.Studies have shown that manganese oxide can play an important role in the soil N cycle as an electron acceptor. The addition of manganese oxide（birnessite） promoted the transformation of NH₄⁺ to NH3 in different p H hydroquinone glycine pure culture, which indicated that manganese oxide has a positive effect on NH3 supply in nitrification. Soil organic matter can also significantly promote nitrification, and it could because the organic matter through the ammonification of ammonia is the source of the substrate of nitrification, moreover the organic matter can be used as carbon source to promote the growth of microbes in soils. These indicated that NH3 produced through the ammonification is an important source in acid soils. Ammonia oxidizing bacteria（AOB） and ammonia oxidizing archaea（AOA） both play a crucial role in the nitrification, as they both have the amo A genes which can produce AMO. The relative contribution of AOB and AOA to nitrification remains controversial.This research aimed to investigate the effects of manganese oxide and soil organic matter on nitrification substrate source of NH3 in acidic soils. Birnessite, acid paddy soil from Chongqing Yubei and acid forest peat soil from Xiaoxing’an Mountains were used as the tested material, the experimental designs are as follows:（1） The exploration of manganese oxide: add birnessite into two acid paddy soils of p H = 4.6 and 4.9, then adjust p H respectively.（2） The exploration of soil organic matter: 1, add glutamic acid, ammonium sulfate into the paddy soil of p H = 4.9, the acetylene treatment as the control group; 2, add acetylene into the acid forest peat soil, and set up the control group; 3, add 0, 1.2, 6.0 mmol N kg-1 ammonium sulfate into the acid forest peat soils, respectively; 4, acid forest peat soil（add 5% neutral paddy soil） as the test soil, add 0, 1.2, 6.0 mmol N kg-1 ammonium sulfate into the soil samples, respectively. All the soil samples were kept at 28 ℃ constant temperature for incubation, and regular sampled for determination of soil nitrate, ammonium, p H, abundances and community structure of the ammonia-oxidizing microorganisms. The results are as follows:（1） Effects of birnessite on nitrification in acid paddy soilNet nitrification rates increased significantly as p H value increase in acid paddy soils, indicating that p H is a limiting factor in the soil nitrification process. After addition of birnessite, the net nitrification rates of the acid paddy soil with p H = 4.6 and 4.9 were decreased from 2.07 and 3.17 mg N kg-1d-1 to 0.60 and 2.71 mg N kg-1d-1, respectively; but the difference in the paddy soil with p H = 4.9 was not significant, which indicated that birnassite did not promote nitrification in this acid soil, but inhibited it, and this may be because the toxicity of manganese oxide on ammonia-oxidizing microorganisms is greater than the positive effects of manganese oxide on transformation of NH₄⁺ to NH3.（2） Effects of organic matter on nitrification in acid soil1, Nitrification in acetylene treated soil did not occur, even negative, and without acetylene processing soil samples were strongly. Since acetylene can inhibit soil nitrification, Showed that soil nitrification is given priority to with autotrophic nitrification. Glutamic acidand ammonium sulfate promotes soil nitrification, but there was no significant difference between the soils with different nitrogen source. Excessive glutamate mineralization, ammonia has no time to be used as nitrogen source.2, Nitrification occurred in acid peat soil while processing of acetylene without nitrification which shows that autotrophic nitrification is still based in this soil. The acid soil, rich in organic matter, was strong mineralization. A significant growth of AOB abundance and carrying out nitrificationhad done in soil samples without acetylene, while AOB, AOA abundance in other processing samples did not change significantly. Therefore, the main contributor to the soil nitrification process may still be AOB.3, Soilsamples processing of Control,1.2,6.0 mmol N kg-1 Ammonium sulfate have carried out intense mineralization and nitrification, and have no significant differences of net nitrification in the concentration gradient of nitrogen, that is, the soil nitrification process with different concentrations of nitrogen the response is the same. Thus, we hypothesized that the main source of the acid peat forest soil nitrification substrates is not exogenous inorganic ammonium nitrogen, but mineralization of soil organic matter produced inorganic nitrogen. During the culture, ammonia-oxidizing microorganisms AOB and AOA abundance increased significantly, but shows no significant difference between the treated soil samples with different concentrations of ammonium sulfate, which suggests that AOB and AOA may have contributed to nitrification and exogenous inorganic nitrogen concentration had no effect on the ammonia-oxidizing microorganisms.4, Acid peat soils after adding neutral paddy soil, and 0, 1.2, 6.0 mmol N kg-1 ammonium sulfate, the net nitrification rates were 1.52, 2.15, 3.30 mg N kg-1, were higher than the original soil sample（1.20 mg N kg-1）, and with the increase of nitrification of ammonium sulfate concentration increased. Nitrification substrates no longer rely solely on the mineralization of organic matter produced ammonia, can also use exogenous inorganic nitrogen. Training process, amo A gene copy number AOB and AOA increased significantly, and ammonium sulfate promote the growth of the AOB. In the incubation, AOB and AOA amo A gene copy number increased significantly; and with the increase of AOB amo A gene copy number of ammonium sulfate concentration increased,which suggests that ammonium sulfate significantly promote the growth of the AOB. Pearson correlation analysis showed, AOB abundance increased in samples treated with control, 0, 1.2, 6.0mmol Nkg-1 ammonium and net nitrification rate were a significant positive correlation, with correlation coefficient r = 0.898（p <0.01, n = 12）, while not AOA（r = 0.418, p = 0.176, n = 12）. After adding neutral soil, changed the soil ammonia-oxidizing bacterial community structure AOB, and community structure of ammonia-oxidizing archaea AOA almost unchanged. The source of nitrification substrate NH3 is changed with Changing of AOB community structure. Those results showed that AOB may dominate the nitrification process in the acid peat soil paddy soil after adding neutral paddy soil.This study investigated the effects of manganese oxide and organic matters on NH3 sources in acid soils. We get the following conclusions: 1, Acid paddy soil and acid peat soil nitrification is given priority to with autotrophic nitrification, and the acid peat soil has strongly mineralization. 2, Training process, NH3 may be derived from organic matter mineralization rather than ammonium sulfate. Both ammonia-oxidizing microorganisms AOB and AOA have contributions to nitrification. 3, Nitrification substrates NH3 not only come from organic nitrogen mineralization, but also from ammonium sulfate in acidic peat soil（added 5% neutral purple soil）; and AOB was dominant in the nitrification process rather than AOA. 4, The source of NH3 during nitrification in acidic peat soil is related with microbial community structure of ammonia oxidation.