2.2 Incubation experiments design
To investigate the effects of biochar additions on soil organic carbon change, soil microcosms were designed and constructed using 1m×1m×1m polypropylene boxes. Biochar materials were prepared from rice straw at 550℃ in anaerobic conditions and contained 422.6 g/kg of carbon, 8.4 g/kg of nitrogen, 2.2 g/kg of phosphorus, and pH of the biochar was 8.34.
Incubation experiments were carried out in the field. The application rates of biochar were set to 0.5% (T0.5), 1% (T1), 2% (T2), 5% (T5), and 10% (T10) by biochar weight to soils, and each treatment was performed with three replications. Soil alone was the control treatment (CK). Soil and biochar materials were fully mixed before they were put into the microcosm box. The soil depth was set to 50 cm. T0.5, T1, and T2 treatment were categorized into the lower level of additions (LK), while T5 and T10 were at a higher level (HK) (Yoo and Kang, 2012).
Incubation boxes were placed in the field without extra water addition but natural precipitation, aiming to simulate natural soil water conditions. Experiments started in May 2018, and finished in November 2018, spanning a growing season in Northeast China. Soil samples were collected monthly for chemical analysis.
2.3 Phospholipid fatty acid (PLFA ) analysis
PLFA extraction and analysis were performed according to the method described by (Zhang, et al., 2012a). In brief, fresh soils were freeze-dried and extracted with a chloroform-methanol-citrate buffer mixture (1:2:0.8). The phospholipids were separated from other lipids on a silicic acid column. Phospholipid phosphate concentration was determined using the spectrometric method. Phospholipids were subjected to a mild-alkali methanolysis, and the resulting fatty acid methyl esters were separated by gas chromatography with a flame ionization detector (Agilent 6890N). The carrier gas was helium, and the temperature increased to 260℃ from 170℃ at a rate of 5℃/min. The inner standard, a mixture of 37 fatty acid methyl ester (FAME), was used to identify and quantify the response of individual fatty acids (Steinbeiss et al., 2009). The PLFA makers used for taxonomic microbial groups were shown in Table 1.