2.4 | laboratory treatment
The collected grazing soil samples of different thicknesses were evenly divided into two parts per replicate, one for analysis and determination of soil nutrient content and the other for determination of soil microorganisms MBC and MBN. After air drying, the soil samples were sieved (0.149 mm) to remove impurities such as plant roots and stones, and the soil was ground after clean removal. Soil organic matter, total soil nitrogen, total soil phosphorus and pH were determined. Soil organic matter was oxidised by the potassium dichromate method, and total soil nitrogen was determined by the Kjeldahl method. Soil pH was determined by acid-base test paper for another fresh soil sample used for soil microbial MBC and MBN detection, soil impurities were removed after sieving, chloroform fumigation method was applied, soil solution was extracted after soil fumigation with chloroform, and soil MBC and MBN were calculated based on the difference between fumigated and non-fumigated soil carbon and nitrogen. K2Cr2O7oxidation method(Murano et al., 2021) is used for soil organic matter. In the presence of excess H2SO4, K2Cr2O7is used to oxidize organic carbon, and the remaining oxidant is dripping back with standard FeSO4. The consumed oxidation dose is used to calculate the content of organic carbon.
The total nitrogen content of the soil is determined by the Kjeldahl method (Song Shuhui et al., 2019). The soil is digested with H2SO4 and a catalyst is added to accelerate the decomposition of organic matter and convert organic nitrogen to ammonia in solution. Finally, the distilled ammonia is titrated with standard acid.
The CHCl3 fumigation culture method is used for soil microbial biomass carbon (MBC). The collected soil samples are sieved to 6.35 mm and then fumigated with chloroform. After fumigation, 10 g of fresh soil is added for reculture. The micro-organisms killed by fumigation in the original soil sample release CO2 after mineralisation by the newly added micro-organisms. The difference between the CO2 released during the culture period and the mineralisation ratio is used to calculate the MBC of the soil.
\begin{equation} E_{c}=\frac{(V_{0}-V_{S})\times C_{FeSO4}\times\frac{12}{4}\times 1000\times\frac{50}{2})}{W_{S}}\nonumber \\ \end{equation}\begin{equation} \text{soil\ microbial\ biomass\ carbon}\left(\text{MBC}\right)=\frac{E_{c}^{CHCL3}-E_{C}^{\text{CK}}}{0.38}\nonumber \\ \end{equation}
(In the formula : Company is (mg/kg),Ec is the amount of organic carbon,V0 and Vsare the volume of solution consumed for titration of blank and soil samples respectively,C FeSO4 is the concentration of FeSO4 solution, 12 is the molar mass of carbon,1000 is the conversion from kg to g, 50 / 2 is 50ml, 2ml is absorbed from 50ml extract, and Ws is the mass of dried soil. 0.38 is the Microbial mineralization rate constant.)
Soil microbial biomass nitrogen (MBN) is extracted by the CHCl3 fumigation leaching method. Fresh soil is fumigated with CHCl3 for 24 h and then extracted with 0.5 mol/L K2SO4 solution. 10 ml of the filtrate is absorbed and placed in the digestion furnace. K2SO4-CuSO4-Se mixed catalyst and 4ml H2SO4 are added. At the same time, 10ml filtrate is added to catalyst and H2SO4 to set 2-3 blanks, after 12h, it is digested in the digestion furnace at 150℃ to remove water, and then digested at 320℃ to clarify. After standing for 2-3 hours, the nitrogen content is determined by semi-micro distillation.
Calculation formula:
\begin{equation} E_{N}=\frac{(V_{s}-V_{0})\times C_{H2SO4}\times 14\times 1000\times\frac{50}{20})}{W_{S}}\nonumber \\ \end{equation}\begin{equation} \text{soil\ microbial\ biomass\ nitrogen}\left(\text{MBN}\right)=\frac{E_{N}^{CHCL3}-E_{N}^{\text{CK}}}{0.45}\nonumber \\ \end{equation}
(In the formula : Company is (mg/kg),EN is the total nitrogen content,V0 and Vsare the volume of solution consumed for titration of blank and soil samples respectively,C H2SO4 is the concentration of H2SO4 solution, 14 is the molar mass of nitrogen ,1000 is the conversion from kg to g, 50 / 20 is 50ml, 20ml is absorbed from 50ml extract, and Ws is the mass of dried soil. 0.45 is the Microbial mineralization rate constant.)