The chloroform fumigation extraction (CFE) method determines microbial biomass carbon (MBC) or nitrogen (MBN) by calculating the increase in extractable carbon (C) or nitrogen (N) due to microbial lysis during chloroform fumigation. In China, many studies have focused on the impacts of N and phosphorus (P) addition on soil MBC and MBN in forest ecosystems, where substantial atmospheric N deposition has strongly acidified soils. The addition of nutrients may alter the extraction process applied in the CFE method, potentially influencing the MBC and MBN determined by the CFE method independently of the actual microbial biomass. In this study, we tested whether the MBC and MBN determined by the CFE method were biased by the experimental addition of N and P in strongly acidified Chinese forest soils by adding N and P to the soils immediately before chloroform fumigation, which should not affect the actual microbial biomass. P addition significantly elevated the dissolved organic carbon (DOC) content, especially after fumigation, while N addition significantly reduced the dissolved nitrogen (DN) content. The added N was subtracted using blank samples without soil. However, the altered DOC and DN contents did not affect the MBC and MBN contents determined by the CFE method. In conclusion, our study suggests that the CFE is a relatively robust method to test the impacts of nutrient addition on microbial biomass in the strongly acidified soils of Chinese forests. We also suggested that: (i) even if a fertilization experiment results in an elevated DOC content following P addition, it does not necessarily indicate a stimulation of DOC production by microbes; and (ii) the soil adsorption capacity or the strength of microbial N uptake during the extraction procedure applied in the CFE method may affect the determination of MBN by influencing the DN extraction efficiency.
The chloroform fumigation extraction (CFE) method (
Researchers have investigated the impacts of nutrient addition, such as nitrogen (N) and phosphorus (P), on soil microbial biomass. This has enabled an evaluation of the impacts of anthropogenic nutrient loading into soils, such as atmospheric nutrient deposition or nutrient fertilization, on soil microbial biomass (
Several studies have reported methodological weaknesses of the CFE method. For example,
However, the robustness of the CFE method for testing the impacts of nutrient addition on soil microbial biomass has not been fully examined. Added nutrients may alter the extraction process applied in the CFE method, potentially influencing the MBC and MBN determined by the method independently of the actual microbial biomass. It is possible that the changes in soil pH through nutrient addition affect the microbial biomass determined by the CFE method, because soil pH can affect the extraction efficiency of DOC by K2SO4 (
The aim of this study was to test whether the MBC and MBN determined by the CFE method are biased by the experimental addition of N and P in strongly acidified Chinese forest soils. By adding N and P immediately before fumigation, we evaluated the CFE method-dependent impacts of nutrient addition on microbial biomass determination. Because the addition of N and P immediately before fumigation should not affect the actual microbial biomass (
Soil samples for the experiment were collected from six forests in China. Three of the six forest sites were located in the Dinghushan Biosphere Reserve (DHS; 23° 10′ N, 112° 10′ E -
Surface soil samples (0-10 cm) were collected from subplots at the six forest sites using soil cores (three, three, and four subplots in DHS, HS, and JGS, respectively). Soil samples taken from the same site were combined, and six replicates were prepared for the experiment. We sieved the soil samples through a 2-mm sieve after removing the fine roots and coarse organic matter. Sieved soil samples (6 g) were placed in bottles (glass bottles for fumigated soils and plastic bottles for unfumigated soils) and 1.5 mL N (500 µg N per 1.5 mL solution in the form of NH4NO3) or P (500 µg P per 1.5 mL solution in the form of KH2PO4) were added. The final concentrations of the added N and P (around 100 µg N and P per g soil) were in a similar range to that of previous laboratory experiments where nutrient concentrations were decided based on the nutrient doses in the field (
A paired
The DOC and DN contents in our study sites ranged from 130 to 300 µg C g soil-1 and from 15 to 45 µg N g soil-1, respectively. Chloroform fumigation caused a large elevation in both the DOC and DN contents (by ~100 µg C g soil-1 and 10 µg N g soil-1, respectively -
N addition did not affect the MBC content determined by the CFE method (
Both the paired
The decrease in the DN content following N addition in both the unfumigated and fumigated soils indicated that a portion of the added N (NH4NO3) was adsorbed by the soil or immobilized by microbes (
We also found that P addition elevated the DOC content (especially DOCaft -
By adding N and P immediately before fumigation, we conducted tests to determine whether the MBC and MBN measured by the CFE method were biased by the experimental addition of N and P in strongly acidified Chinese forest soils. Our results demonstrated that the MBC and MBN contents were not significantly biased. Despite the increase in the DOC content following P addition (
By testing whether the N or P addition immediately before fumigation affected the results of a microbial biomass determination by the CFE method, we evaluated the robustness of the CFE method for determining the impacts of N and P addition on microbial biomass C and N in the strongly acidified soils of Chinese forests. We found that P addition significantly elevated the DOC content (especially DOCaft); N addition significantly reduced the DN content; and the altered DOC and DN contents did not change the MBC or MBN contents determined by the CFE method. We concluded that CFE is a relatively robust method to determine the impacts of nutrient addition on microbial biomass in the strongly acidified soils of Chinese forests. The results also suggest that even if a fertilization experiment revealed an elevated DOC content after P addition, it does not necessarily indicate DOC production by microbes. The soil adsorption capacity or strength of microbial N uptake during the extraction procedure applied in the CFE method may affect the determination of MBN by influencing the DN extraction efficiency.
TM conceived the research and wrote the draft of the manuscript, TM, SW, and CW performed experiment, JM and WZ established research sites, all of the authors joined the discussion of the research. We thank Mr Fu and Ms Hu for their support for our field work. This study was financially supported by National Natural Science Foundation of China (no. 42077311, no. 41731176), Grant-in-Aid for JSPS Postdoctoral Fellowships for Research Abroad (28-601), and a grant from the Sumitomo Foundation (153082).
Effects of experimental (a) N addition and (b) P addition on the dissolved organic carbon (DOC) content before and after chloroform fumigation. The DOC was extracted by 0.5 M K2SO4 after 30 min of shaking. Error bars indicate the standard error of data from six sites. Statistical significance was determined by a paired
Effects of experimental (a) N addition and (b) P addition on the dissolved nitrogen (DN) content before and after chloroform fumigation. The DN was extracted by 0.5 M K2SO4 after 30 min of shaking. Error bars indicate the standard error of data from six sites. Statistical significance was determined by a paired
Effects of experimental (a) N addition and (b) P addition on the microbial biomass carbon (MBC) content before and after chloroform fumigation. Error bars indicate the standard error of data from six sites. A paired
Effects of experimental (a) N addition and (b) P addition on the microbial biomass nitrogen (MBN) content before and after chloroform fumigation. Error bars indicate the standard error of data from six sites. A paired
Effects of experimental (a) N addition and (b) P addition on soil pH. Error bars indicate the standard error of data from five sites (BF, MF, PM, AA, and EU). A paired
Selected basic characteristics (mean ± standard error) of the six forest sites investigated. Data collection and source: (a) measured in 2015 (from
Characteristics | Sites | |||||
---|---|---|---|---|---|---|
BF | MF | PM | AA | EU | MDF | |
Soil organic C (g kg-1) | 40.0 ± 4.2 a | 32.1 ± 4.0 b | 23.3 ± 1.6 b | 23.8 ± 1.7 c | 18.5 ± 0.4 c | 63 ± 13 d |
Soil total N (g kg-1) | 2.9 ± 0.5 a | 2.0 ± 0.3 b | 1.4 ± 0.2 b | 2.0 ± 0.1 c | 1.5 ± 0.2 c | 3.4 ± 0.5 d |
Soil available P (mg kg-1) | 2.1 ± 0.4 a | 1.0 ± 0.3 b | 1.5 ± 0.3 b | 2.5 ± 0.2 c | 2.1 ± 0.1 c | 6.9 ± 2.0 d |
DOC (µg C g-1) | 220.7 ± 3.6 e | 154.4 ± 2.5 e | 208.0 ± 4.4 e | 275.6 ± 3.5 e | 297.9 ± 3.6 e | 135.0 ± 2.0 e |
DN (µg C g-1) | 29.9 ± 1.9 e | 17.9 ± 1.0 e | 25.9 ± 1.8 e | 38.5 ± 1.1 e | 41.8 ± 2.2 e | 25.4 ± 0.8 e |
Soil pH (H2O) | 3.53 f | 3.68 f | 3.6 f | 3.4 f | 3.65 f | 4.25 ± 0.10 d |
Soil water content | 0.24 f | 0.18 f | 0.22 f | 0.23 f | 0.27 f | 0.13 f |