石灰性紫色土硝化作用及硝化微生物对不同氮源的响应

Response of Nitrification and Nitrifiers to Different Nitrogen Sources in an Alkaline Purple Soil

土壤中发生的硝化作用是对p H高度敏感的典型过程。本文采用室内恒温培养法,结合定量PCR和高通量测序,研究石灰性紫色土硝化作用以及氨氧化细菌(Ammonia-oxidizing bacteria,AOB)、氨氧化古菌(Ammonia-oxidizing archaea,AOA)、亚硝酸盐氧化细菌(Nitrite-oxidizing bacteria,NOB)的丰度与群落结构对不同氮源的响应。结果表明:不同氮源均刺激土壤硝化作用的发生,CO(NH2)2处理下的净硝化速率最大,约是CK处理的4.76倍,(NH_4)2SO4和NH_4Cl处理下的净硝化速率分别为N 3.88和3.34 mg kg-1d-1。相比于(NH_4)2SO4和CO(NH2)2处理,NH_4Cl处理降低了硝态氮的累积量,抑制了铵态氮的减少量。AOB amo A基因拷贝数在28 d培养过程中变化显著(p<0.05),在(NH_4)2SO4和CO(NH2)2处理中呈先增长后降低趋势,在NH_4Cl处理中呈持续增长趋势;而AOA amo A基因拷贝数无显著变化(p>0.05)。说明石灰性紫色土硝化作用的主要推动者是AOB,而不是AOA。在28 d培养过程中,亚硝酸盐氧化细菌占总微生物的比例高于氨氧化细菌和古菌,意味着石灰性紫色土中可能存在全程氨氧化微生物(Comammox)。高通量测序的结果表明:石灰性紫色土中AOB的优势种群为亚硝化螺菌Nitrosospira Cluster 3,AOA的优势种群是土壤古菌Group 1.1b,NOB的优势种群是硝化螺菌Nitrospira。

【Objective】Nitrification in soil is a highly sensitive process to pH. Responses of nitrification rates and the community structures of nitrifying microorganisms to different N sources in an alkaline purple soil were studied to elucidate the microbiological mechanisms for nitrification. 【Method】Three different N sources and the blank control were used in the 4-week incubation study. Net nitrification rate was calculated by the differences of nitrate concentrations at day 0 and 28. The amoA gene abundances for ammonia oxidizing bacteria（AOB） and ammonia oxidizing archaea（AOA） were measured by quantitative PCR before and after the incubation.The relative abundance of nitrite oxidizing bacteria（NOB）was analyzed by binning the sequences of the 16 S rRNA gene and the amoA gene into operational taxonomic unit（OTUs） at 97% similarity level.The changes of community structures forAOA, AOB and NOB were studied by high-throughput sequencing method before and after the incubation.【Result】In the 4-week incubation study,compared with blank control（CK）, soil nitrification rate was stimulated by application of all three kind of nitrogen sources：（（NH4）2 SO4, NH4 Cl and CO（NH2）2）. The net nitrification rate for blank control（CK） was N 0.86 mg N kg^-1 d^-1. The highest net nitrification rate was observed for CO（NH2）2 treatment（N3.88 mg kg^-1 d^-1）, which was more than 4-times higher than CK. The addition of NHand（NH4）2 SO4, showed similar stimulation on nitrification to CO（NH2）2 application. The net nitrification for NH4 Cl and（NH4）2 SO4 were N 3.34 and 3.88 mg kg^-1 d^-1, respectively. But, NH4 Cl also reduced the accumulation of nitrate and inhibited the reduction of ammonium when compared with（NH4）2 SO4, and CO（NH2）2. Along with the accumulation of nitrate, the copies of amoA gene in ammonia oxidizing bacteria（AOB） increased significantly during the first two weeks of incubation（p0.05）. The copy numbers of bacterial amoA genes increased from 0.88×10^7 g^-1 soil and 0.85×10^7 g^-1 soil at day-0 to3.38×10^7 g^-1 soil and3.55×10^7 g^-1 soilat the day 14 of the incubation, then decreased to 1.46×10^7 g^-1 soil and 1.69×10^7 g^-1 soil at the day-28, for（NH4）2 SO4, and CO（NH2）2 treatments respectively.The copy numbers of bacterial amoA genes were significantly lower in NH4 Cl treatment than（NH4）2 SO4, and CO（NH2）2 addition at the day 14（p〉0.05）.On the other hand, the copies of amoA for AOA did not change significantly during incubation（p0.05）.Results indicated that nitrification in alkaline purple soil was mainly driven by AOB, but not AOA.Pyrosequencing of the 16 S rRNA genes was performed at the whole microbial community level for different treatments and control before and after incubation. Approximately more than 30 000 high-quality 16 S rRNA reads were obtained, and targeted reads from putative AOA, AOB and NOB sequences were selected for subsequent analysis. The high-throughput sequencing results further showed that the dominant nitrifying microorganisms were mainly related toNitrospira, Nitrososmonas and Nitrosospira in the alkaline purple soil. The dominant AOBwere classified into Nitrosospira Cluster 3, and the dominant AOA were affiliated with Group 1.1 b. Furthermore, the relative abundance of NOBwas much higher than that of AOB and AOA,which may imply the presence of Comammox in the studied alkaline purple soil.【Conclusion】Results showed that the nitrification in alkaline purple soil was stimulated by the addition of（NH4）2 SO4,NH4 Cl and CO（NH2）2. ButNH4 Cl also showed a inhibition effect on nitrification when compared with（NH4）2 SO4, and CO（NH2）2 application. The nitrification process in alkaline purple soil was mainly driven by AOB, but not AOA. The evidences from the high-throughput sequencing results further indicated that the dominant population of ammonia oxidizing bacteria was classified into Nitrosospira Cluster 3 in the alkaline purple soil, and AOA was mainly the Group 1.1 b. For NOB, Nitrospira was the dominatedspecies.