Okano Yutaka, Hristova Krassimira R, Leutenegger Christian M, Jackson Louise E, Denison R Ford, Gebreyesus Binyam, Lebauer David, Scow Kate M
Department of Land, Air, and Water Resources, University of California, Davis, California 95616, USA.
Appl Environ Microbiol. 2004 Feb;70(2):1008-16. doi: 10.1128/AEM.70.2.1008-1016.2004.
Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 x 10(5) cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH4)2SO4 application. AOB populations in amended treatments increased from an initial density of approximately 4 x 10(6) cells/g of dry soil to peak values (day 7) of 35 x 10(6) and 66 x 10(6) cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 x 10(9) and 2.2 x 10(9) cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 x 10(6) to 38.0 x 10(6) cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH4+ h(-1) cell(-1) and decreased with time in both microcosms and the field. Growth yields were 5.6 x 10(6), 17.5 x 10(6), and 1.7 x 10(6) cells/mol of NH4+ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.
自养型氨氧化细菌（AOB）进行的铵氧化是农业和自然生态系统中的一个关键过程，对全球有着重大影响。过去，由于这些微生物极难培养，人们对AOB的生态学和生理学了解不足。分子技术的最新应用增进了我们对AOB的认识，但基于PCR技术的必要性使得定量测量变得困难。针对氨单加氧酶基因（amoA）部分序列开发了一种定量实时PCR检测方法，用于估计土壤中AOB的种群数量。该检测方法的检测限为每克干土1.3×10⁵个细胞。通过施加0、1.5或7.5 mM硫酸铵，在土壤微观系统中测量铵浓度对AOB种群密度的影响。施加（NH₄）₂SO₄后28天监测AOB种群数量以及铵和硝酸盐浓度。在改良处理中，AOB种群数量从初始密度约每克干土4×10⁶个细胞分别增加到1.5 mM和7.5 mM处理中第7天的峰值，即每克干土35×10⁶和66×10⁶个细胞。无论氨浓度如何，总细菌的种群数量（通过通用细菌探针的实时PCR定量）保持在每克土壤0.7×10⁹至2.2×10⁹个细胞之间。在一块番茄田地块上进行了施肥实验，以测试在微观系统中观察到的AOB密度变化是否也能在田间检测到。到第39天，AOB种群数量从每克土壤8.9×10⁶个细胞增加到38.0×10⁶个细胞。在微观系统实验中，1.5 mM和7.5 mM处理的世代时间分别为28小时和52小时，在田间研究的铵处理中为373小时。每个细胞的估计氧化速率最初在0.5至25.0 fmol NH₄⁺ h⁻¹ cell⁻¹之间，并在微观系统和田间均随时间下降。在1.5 mM和7.5 mM微观系统处理以及田间研究中，生长产量分别为每摩尔NH₄⁺产生5.6×10⁶、17.5×10⁶和1.7×10⁶个细胞。在第二项田间实验中，即使在测量前8个月最后一次施用铵肥，每年施肥的土壤中AOB种群数量仍显著高于未施肥土壤，这表明铵肥对AOB种群数量有长期影响。
