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.)