Quantification of Ammonia-Oxidizing Bacteria in Arable Soil by Real-Time PCR

Abstract

Real-time PCR was used to quantify populations of ammonia-oxidizing bacteria representing the ␤ subdivision of the class Proteobacteria in samples of arable soil, both nitrogen fertilized and unfertilized, from Mellby, Sweden. Primers and probes targeting a 16S ribosomal DNA region of the ammonia-oxidizing bacteria were designed and used. In the fertilized soil there were ϳ6.2 ؋ 10 7 ammonia-oxidizing bacteria per g of soil, three times more than the number of bacteria in the unfertilized soil. The lytic efficiency of bead beating in these soils was investigated by using populations of free or loosely attached bacteria, bacteria tightly bound to particles, and bacteria in nonfractionated samples. The shapes of the curves generated in these tests showed that the concentration of template DNA released at various times remained constant after 10 to 100 s of bead beating.Quantification of microbial populations is important in many aspects of microbial ecology. The development of molecular biological methods involving PCR has led to new techniques that are not limited by the culturability of the microorganisms. Since it is known that only a small proportion of the bacteria in soils can be cultivated under standard laboratory conditions, the PCR-based quantification methods have found many applications. Three PCR-based methods, limiting-dilution PCR (27, 31), kinetic PCR (3, 14), and competitive PCR (10, 11, 23), have been used for quantitative analysis of DNA having different origins. However, kinetic PCR and limitingdilution PCR often have the disadvantage of relying on endpoint measurements of the amount of DNA produced, which makes it difficult to deduce the initial concentration of template DNA. In competitive PCR it may be difficult to achieve the same affinity of the primers for the target and competitive molecules, which complicates quantification. A modified PCR technique, real-time PCR (13), measures the DNA concentration continuously during amplification, which enables the initial template concentration to be determined and the cell numbers to be more accurately deduced without the use of a competing molecule.Quantification of ammonia-oxidizing bacteria (AOB), which are responsible for the oxidation of ammonia to nitrite in the nitrification process, has been attempted by using several different methods. These include the most-probable-number technique (6,7,19), in situ hybridization (29), a competitive enzyme-linked immunosorbent assay using monoclonal antibodies (28), and competitive PCR (17, 26, 30) based on traditional methods of amplification. However, all of these methods have significant disadvantages. Thus, a reliable and reproducible method for quantifying AOB would be valuable for evaluating correlations between microbial activities and cell numbers, the effects of different treatments on cell density, and population changes in time and space. Determination of DNA concentrations with real-time PCR overcomes some of the problems associated with traditional PCR. The real-time PCR technique is based ...

Ammonia-oxidizing bacteria respond to multifactorial global change

“…This is consistent with our observations; we observed a statistically significant effect only when both CO 2 and water were elevated. Several studies have also reported significant increases in AOB abundance in response to nitrogen fertilization (21,27,28), an effect we did not observe in our system. However, those studies were designed to mimic agricultural fertilization, with nitrogen applied at much higher rates than in our study.…”

Structure and composition of bacterial and fungal community in soil under soybean monoculture in the Brazilian Cerrado

“…But, additional caution should accompany these results because the data were obtained from relatively few composite samples. Besides the DGGE bias already discussed, the impacts of DNA extraction and PCR amplification may have also contributed to further bias (20). Therefore, further research is needed to confirm the observations reported herein.…”

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

“…Although sample enrichment does increase sensitivity, it essentially renders RT-PCR non-quantitative. RT-PCR has also been used to quantify ammonia-oxidizing bacteria in soil (Hermansson and Lindgren 2001), enterococci and human adenovirus in water (He and Jiang 2005), Vibrio vulnificus in shellfish and water (Panicker et al 2004), Lactobacillus salivarius in broiler chickens (Harrow et al 2007) and Clostridium tyrobutyricum in milk (López-Enríquez et al 2007).…”