2.5 Measurement of CO2 efflux rate and microbial activity as qCO2
The CO2 efflux rate was measured by a closed-chamber system (Seki et al., 2019) at a frequency of approximately every 2 week in the rainy season and every month in the dry season for a total of 40 times throughout the experimental period. Polyvinyl chloride (PVC) columns (diameter 13 cm, height 30 cm) were inserted randomly in each plot at the end of each September or August, i.e., after FYM application. We waited at least 1 week after the installation until measuring the CO2 efflux rate, so as not to disturb the plots when installing columns. Columns were re-installed within a plot every year, as mentioned above. Since soil respiration is composed of microbial respiration and plant-root respiration, plant-root respiration was excluded by the trenching method (Shinjo et al., 2006), following the detailed in Seki et al (2019). Gases were sampled at 0 min and again 40 min after the top of the column was covered with a plastic sheet, and analyzed with an infrared CO2 analyzer (ZFP9-AA11; Fuji Electric, Japan) equipped with a voltage capture detector (C-R8A; Shimadzu, Japan) and N2 carrier gas (Shinjo et al., 2006). The CO2 efflux rate was calculated based on the increase in CO2 concentration in the column after 40 min. Two columns were installed in each plot, and we used the average data in each plot with three replicates (plots). The CO2efflux rate was always measured between 08:00 and 11:00 am in the field.
To evaluate the microbial activity as qCO2 (generally termed as a metabolic quotient) (Anderson & Domsch, 1985), we divided the measured CO2 efflux rate by the MBC. In the calculation, both CO2 efflux rate and MBC were expressed on an area basis (µg CO2-C m−2h−1 and mg MBC m−2, respectively).