10:45 〜 12:15
[AHW18-P23] Relationship between inorganic nitrogen and soil bacteria in four weeks after application of cattle compost
キーワード：無機態窒素、土壌、牛ふん堆肥
The utilization of nitrogen fertilizer and compost in agricultural land is known to enhance crop yield; however, this practice can lead to an increase in nitrogen concentration in groundwater due to the surplus seeping underground. To ensure the sustainable use of groundwater resources and promote recycle-oriented agriculture, an accurate estimation of the amount of nitrogen mineralized from compost and the determination of the appropriate amount of fertilizer to be applied are necessary.
In recent years, genetic analysis methods, such as quantitative polymerase chain reaction (qPCR), have been employed for detecting microorganism abundance in the environment. However, the use of qPCR in compost and Okinawan soils has been limited. Therefore, this study conducted an incubation experiment, based on the heat-retention method, using soil and cattle manure from the southern region of Okinawa Island to investigate the relationship between the amount of inorganic nitrogen and the number of soil bacteria in the initial stage of compost application by quantifying the 16S rRNA gene using qPCR.
To conduct the experiment, Okinawan soil (also known as jargal) and cattle manure were collected from Yaese Town, air-dried, and passed through a 2 mm sieve. Sterilized petri dishes containing ten grams of the samples (adjusted to have 0.05 g of nitrogen from fertilizer) were prepared, and sterile water was added to reach maximum water content of 60% per dry soil. The petri dishes were incubated at 30°C for 4 weeks with weekly measurements of the inorganic nitrogen (NH4+, NO2-, NO3-) concentrations and 16S rRNA gene quantification.
Inorganic nitrogen concentration was measured by transferring 10 g of the culture sample to a 100 ml polyethylene bottle, adding 50 g of water, shaking for 1 hour, filtering through a 0.2 µm filter, and measuring the concentration using ion chromatography (Thermo Aquion). The culture samples were subjected to DNA extraction using FastDNA SPIN Kit for Soil (Q-BioGene), and the 16S rRNA genes were quantified using real-time PCR (Roche LightCycler 480).
The results showed that for inorganic nitrogen in extracted water from fertilizer-supplemented culture samples, the NH4+ concentration exhibited a maximum value of 680 mg/L at week 0 and decreased thereafter. The NO2- concentration increased until week 3, reaching a maximum value of 287 mg/L, and then declined. The NO3- concentration increased significantly during the third and fourth weeks, with a maximum value of 764 mg/L at week 4. The 16S rRNA gene level in the soil reached a maximum of 3.94×109 copies/g of sample at week 2 and subsequently decreased in the sample with the addition of chemical fertilizer.
For the compost-incubated samples, both NH4+ and NO2- concentrations reached a maximum of 11 mg/L and 17.1 mg/L, respectively, at week 1, and then decreased. The maximum value of 364.5 mg/L was reached at week 4. The 16S rRNA gene level in the soil showed a maximum value of 2.60×1010 copies/g of sample at week 3 and then decreased. Furthermore, the 16S rRNA gene content of compost was higher in all weeks, with a 3 to 10-fold difference compared to chemical fertilizer.
These findings suggest that the application of compost to Okinawan soil increases the number of soil bacteria, leading to the decomposition of organic matter in the soil and an increase in inorganic nitrogen (NO3-).
In recent years, genetic analysis methods, such as quantitative polymerase chain reaction (qPCR), have been employed for detecting microorganism abundance in the environment. However, the use of qPCR in compost and Okinawan soils has been limited. Therefore, this study conducted an incubation experiment, based on the heat-retention method, using soil and cattle manure from the southern region of Okinawa Island to investigate the relationship between the amount of inorganic nitrogen and the number of soil bacteria in the initial stage of compost application by quantifying the 16S rRNA gene using qPCR.
To conduct the experiment, Okinawan soil (also known as jargal) and cattle manure were collected from Yaese Town, air-dried, and passed through a 2 mm sieve. Sterilized petri dishes containing ten grams of the samples (adjusted to have 0.05 g of nitrogen from fertilizer) were prepared, and sterile water was added to reach maximum water content of 60% per dry soil. The petri dishes were incubated at 30°C for 4 weeks with weekly measurements of the inorganic nitrogen (NH4+, NO2-, NO3-) concentrations and 16S rRNA gene quantification.
Inorganic nitrogen concentration was measured by transferring 10 g of the culture sample to a 100 ml polyethylene bottle, adding 50 g of water, shaking for 1 hour, filtering through a 0.2 µm filter, and measuring the concentration using ion chromatography (Thermo Aquion). The culture samples were subjected to DNA extraction using FastDNA SPIN Kit for Soil (Q-BioGene), and the 16S rRNA genes were quantified using real-time PCR (Roche LightCycler 480).
The results showed that for inorganic nitrogen in extracted water from fertilizer-supplemented culture samples, the NH4+ concentration exhibited a maximum value of 680 mg/L at week 0 and decreased thereafter. The NO2- concentration increased until week 3, reaching a maximum value of 287 mg/L, and then declined. The NO3- concentration increased significantly during the third and fourth weeks, with a maximum value of 764 mg/L at week 4. The 16S rRNA gene level in the soil reached a maximum of 3.94×109 copies/g of sample at week 2 and subsequently decreased in the sample with the addition of chemical fertilizer.
For the compost-incubated samples, both NH4+ and NO2- concentrations reached a maximum of 11 mg/L and 17.1 mg/L, respectively, at week 1, and then decreased. The maximum value of 364.5 mg/L was reached at week 4. The 16S rRNA gene level in the soil showed a maximum value of 2.60×1010 copies/g of sample at week 3 and then decreased. Furthermore, the 16S rRNA gene content of compost was higher in all weeks, with a 3 to 10-fold difference compared to chemical fertilizer.
These findings suggest that the application of compost to Okinawan soil increases the number of soil bacteria, leading to the decomposition of organic matter in the soil and an increase in inorganic nitrogen (NO3-).