Rainer Georg Joergensen

The fumigation-extraction method to estimate soil microbial biomass: Calibration of the kEC value

Abstract The kEC value (=extractable part of microbial biomass C) of the fumigation-extraction (FE) method was assessed on the basis of 153 soils (94 arable, 46 grassland and 13 forest soils) by indirect calibration using the fumigation-incubation (FI) method. Sixty-six soils were investigated for the first time and the data on a further 87 soils were obtained from the literature. The single kEC values ranged from 0.23 to 0.84. A split according to the form of land use resulted in a significantly (Scheffe, P = 0.05) lower kEC value for the arable soils (0.42; n = 94) in comparison to those for the grassland (0.49; n = 46) and the forest soils (0.51; n = 13). This difference is mainly due to the significant effects of the respiration rate measured in non-fumigated control samples of the FI method which was used for calibration of the kEC value. For that reason, I investigated the effects of incubation temperature (22°, 25° and 28°C) on biomass C data obtained by the FI method, and thus on the kEC value of the FE method, and discuss further problems of direct and indirect calibration. Based on experimental and literature data, I conclude that the kEC values of Vance et al. (Soil Biology & Biochemistry19, 703–707, 1987) and Wu et al. (Soil Biology & Biochemistry22, 1167–1169, 1990) remain valid. A kEC value of 0.38 can be recommended for C analysis by dichromate consumption and a kEC value of 0.45 for that by UV-persulfate or oven oxidation.

Relationship between SIR and FE estimates of microbial biomass C in deciduous forest soils at different pH

Two methods to estimate soil microbial biomass, viz. substrate-induced respiration (SIR) and fumigation-extraction (FE), were compared using 40 beech (Fagus sylvatica L.) forest soils. Soil chemical properties, microbial biomass and activity indices differed over a wide range but generally fitted a normal distribution. Microbial biomass C estimates by SIR and FE were significantly correlated with r = 0.94. Microbial biomass C was significantly correlated with soil pH, soil organic C, K2SO4-extractable C and especially with the basal respiration rate, irrespective of the method used. As expected from the basic principles of an extraction and a physiological method, FE was more affected than was SIR by soil organic matter, with SIR being more affected by soil pH and basal respiration. The development of different microbial community structures at different pH values affects the SIR and FE methods in reverse directions, increasing the differences between the results of the two methods. Estimates of microbial biomass C-to-soil organic C ratio by SIR and FE were significantly interrelated, with r = 0.89 (P < 0.0001) and were closely connected with soil pH. Estimates of the metabolic quotient ent qCO2 by SIR and FE were also significantly correlated, but with r = only 0.54 (P < 0.001).

Carbon and nitrogen relationships in the microbial biomass of soils in beech (Fagus sylvatica L.) forests

Soils from 38 German forest sites, dominated by beech trees (Fagus sylvatica L.) were sampled to a depth of about 10 cm after careful removal of overlying organic layers. Microbial biomass N and C were measured by fumigation-extraction. The pH of the soils varied between 3.5 and 8.3, covering a wide range of cation exchange capacity, organic C, total N, and soil C:N values. Maximum biomass C and biomass N contents were 2116 μg C m-2 and 347 μg N m-2, while minimum contents were 317 and 30 μg m-2, respectively. Microbial biomass N and C were closely correlated. Large variations in microbial biomass C:N ratios were observed (between 5.4 and 17.3, mean 7.7), indicating that no simple relationship exists between these two parameters. The frequency distribution of the parameters for C and N availability to the microflora divided the soils into two subgroups (with the exception of one soil): (1) microbial: organic C>12 mg g-1, microbial:total N>28 mg g-1 (n=23), a group with high C and N availability, and (2) microbial:organic C≤12 mg g-1, microbial:total N≦28 mg g-1 (n=14), a group with low C and N availability. With the exception of a periodically waterlogged soil, the pH of all soils belonging to subgroup 2 was below 5.0 and the soil C:N ratios were comparatively high. Within these two subgroups no significant correlation between the microbial C:N ratio and soil pH or any other parameter measured was found. The data suggest that above a certain threshold (pH 5.0) microbial C:N values vary within a very small range over a wide range of pH values. Below this threshold, in contrast, the range of microbial C:N values becomes very large.

Activity and biomass of soil microorganisms at different depths

We measured microbial biomass C and soil organic C in soils from one grassland and two arable sites at depths of between 0 and 90 cm. The microbial biomass C content decreased from a maximum of 1147 (0–10 cm layer) to 24 μg g-1 soil (70–90 cm layer) at the grassland site, from 178 (acidic site) and 264 μg g-1 soil (neutral site) at 10–20 cm to values of between 13 and 12 μg g-1 soil (70–90 cm layer) at the two arable sites. No significant depth gradient was observed within the plough layer (0–30 cm depth) for biomass C and soil organic C contents. In general, the microbial biomass C to soil organic C ratio decreased with depth from a maximum of between 1.4 and 2.6% to a minimum of between 0.5 and 0.7% at 70–90 cm in the three soils. Over a 24-week incubation period at 25°C, we examined the survival of microbial biomass in our three soils at depths of between 0 and 90 cm without external substrate. At the end of the incubation experiment, the contents of microbial biomass C at 0–30 cm were significantly lower than the initial values. At depths of between 30 and 90 cm, the microbial biomass C content showed no significant decline in any of the four soils and remained constant up to the end of the experiment. On average, 5.8% of soil organic C was mineralized at 0–30 cm in the three soils and 4.8% at 30–90 cm. Generally, the metabolic quotient qCO2 values increased with depth and were especially large at 70–90 cm in depth.

Microbial biomass phosphorus in soils of beech (Fagus sylvatica L.) forests

Thirty-eight soils from forest sites in central Germany dominated by beech trees (Fagus sylvatica L.) were sampled to a depth of about 10 cm after careful removal of the overlying organic layers. Microbial biomass P was estimated by the fumigation — extraction method, measuring the increase in NaHCO3-extractable phosphate. The size of the microbial P pool varied between 17.7 and 174.3 μg P g-1 soil and was on average more than seven times larger than NaHCO3-extractable phosphate. Microbial P was positively correlated with soil organic C and total P, reflecting the importance of soil organic matter as a P source. The mean microbial P concentration was 13.1% of total P, varying in most soils between 6 and 18. Microbial P and microbial C were significantly correlated with each other and had a mean ratio of 14.3. A wide (5.1–26.3) microbial C: P ratio indicates that there is no simple relatinship between these two parameters. The microbial C: P ratio showed strong and positive correlations with soil pH and cation exchange capacity.

Changes in microbial biomass and P fractions in biogenic household waste compost amended with inorganic P fertilizers.

The present study was conducted to evaluate the changes in microbial biomass indices (C, N, and especially P) and in P fractions in compost amended with inorganic P fertilizers. In the non-amended control, the average contents of microbial biomass C, N, and P were 1744, 193, and 63 microg g(-1) compost, respectively. On average, 1.3% of total P was stored as microbial biomass P. The addition of KH(2)PO(4) and TSP (triple super phosphate) led to immediate significant increases in microbial biomass C, N, and P. Approximately, 4.6% of the added TSP and 5.8% of the added KH(2)PO(4) were incorporated on average into the microbial biomass throughout the incubation. Approximately, 4.7% of the 1mg and 5.8% of the 2mg addition rate were incorporated on average into the microbial biomass. In the amendment treatments, the average contents of microbial biomass C, N, and P declined by 44%, 64%, and 49%, respectively. Initially, the average size of the P fractions in the non-amended compost increased in the order (% of total P in brackets) resin P (0.7%)<NaOH-extractable P(i) (inorganic P, 3.0%)<NaOH-P(o) (organic P, 6.9%)<NaHCO(3)-P(i) (11.9%)<NaHCO(3)-P(o) (17.0%)<residual P (24.6%)<HCl-P (35.7%). Initially, the relative contributions of the P fractions in the amended compost treatments increased in the order: NaOH-P(i) (1.0%)<resin P (2.5%)<NaOH-P(o) (4.5%)<NaHCO(3)-P(o) (4.9%)<residual P (14.8%)<HCl-P (15.2%)<NaHCO(3)-P(i) (57.1%). At the end of the 56-day incubation, the largest and highly soluble fraction of NaHCO(3)-extractable P(i) had decreased in place of the less soluble fractions NaOH-extractable P(i) and P(o), but especially HCl-P, but not in place of the insoluble fraction of residual P. The microbial biomass is able to rapidly store significant amounts of easily soluble P and to prevent it from adsorption or other fixation processes.

The determination of δ13C in soil microbial biomass using fumigation-extraction

Abstract The determination of the isotopic composition of the microbial biomass C in soil is an important tool to study soil microbial ecology and the decomposition and microbial immobilization of soil organic C. We discuss advantages and disadvantages of different methods to determine 13C/12C in soil microbial biomass and propose a new procedure that is based on the UV-catalyzed liquid oxidation of fumigated and non-fumigated soil extracts combined with trapping of the released CO2 in liquid nitrogen and subsequent determination of δ 13 CO 2 -C by a gas chromatograph connected with an isotope ratio mass spectrometer (IRMS). This method was evaluated using test solutions with known isotopic composition and soil extracts. Additionally, the method was compared with an off-line sample preparation technique combined with isotope analysis by a dual-inlet IRMS and an on-line analysis using an elemental analyser connected with an IRMS. All methods applied obtained comparable results and there were no significant differences between the δ 13 C values measured. The off-line preparation procedure had the highest precision but it was also the most labour-intensive. The choice of the most suitable method depends mainly on the number of samples that have to be analysed, the salt concentration of the extracts and the differences of δ 13 C that have to be detected. The application of this method with liquid oxidation and subsequent GC-IRMS analysis showed that microbial biomass C of a grassland soil was 13C-enriched by 2‰ δ 13 C PDB compared with the total soil organic C. The addition of maize straw resulted in a rapid immobilization of maize C in the microbial biomass.

Response of soil microorganisms to the addition of carbon, nitrogen and phosphorus in a forest Rendzina

Abstract Soil microorganisms are believed to be controlled by energy and nutrient availability. To evaluate this hypothesis, an experiment was carried out to assess the effects of adding C, N and P on the relationship of active and total microbial biomass by comparing biomass C estimates using substrate-induced respiration (SIR) and fumigation extraction (FE). Effects on the biomass C-to-N and C-to-P ratios were also studied. In a beech forest soil, 110 g C m−2 (glucose), 4 g N m−2 (NH4NO3) and 0.3 g P m−2 (NaH2PO4) were added separately or in combinations for 15 months at biweekly (14 d) intervals. After this period, measurements were taken of soil C and N, basal respiration, biomass C by SIR and biomass C, N and P by FE in the L horizon (litter layer) and in two layers of the A horizon (mineral soil). Microbial properties in litter differed markedly from those in soil, the ratios of biomass CSIR-to-CFE, biomass CFE-to-N and CFE-to-P in litter exceeding those in soil. The differences within the A horizon were small. Total N was significantly increased in the L horizon by the addition of C, N and P. Water content and basal respiration rate were significantly increased by the addition of C, organic C was decreased by the addition of P in the two layers of the A horizon. The addition of C and P caused a significant increase in biomass N, biomass CFE and biomass CSIR, and a significant decrease in the biomass CSIR-to-CFE ratio in all three layers. In contrast, the addition of N caused a significant increase in biomass CSIR only. The addition of P generally caused a significant decrease in the biomass CFE-to-P ratio, but an increase in biomass P only in the L horizon. Although many effects of our treatments were significant, they were relatively small compared to the large amounts of C, N and P added which were many times greater than the annual input. We discussed extensively the observation that the soil and its microflora preserved its original characteristics. The differences between the biomass estimates of SIR and FE are discussed and attributed to changes in the fungal community structure.

Effects of fertilizer and spatial heterogeneity in soil pH on microbial biomass indices in a long-term field trial of organic agriculture.

In the Darmstadt long-term fertilization trial, the application of composted cattle farmyard manure without (CM) and with (CMBD) biodynamic preparations was compared to mineral fertilization with straw return (MIN). The present study was conducted to investigate the effects of spatial variability, especially of soil pH in these three treatments, on soil organic matter and soil microbial biomass (C, N, P, S), activity (basal CO2 production and O2 consumption), and fungal colonization (ergosterol). Soil pH was significantly lower in the MIN treatments than in the organic fertilizer treatments. In the MIN treatments, the contents of soil organic C and total N were also significantly lower (13% and 16%, respectively) than those of the organic fertilizer treatments. In addition, the total S content increased significantly in the order MIN < CM < CMBD. The microbial biomass C content was significantly lower (9%) in the MIN treatments than in the organic fertilizer treatments. Microbial biomass N and biomass P followed microbial biomass C, with a mean C/N ratio of 7.9 and a mean C/P ratio of 23. Neither the microbial biomass C to soil organic C ratio, the metabolic quotient qCO2, nor the respiratory quotient (mol CO2/mol O2) revealed any clear differences between the MIN and organic fertilizer treatments. The mean microbial biomass S content was 50% and the mean ergosterol content was 40% higher in the MIN treatments compared to the organic fertilizer treatments. The increased presence of saprotrophic fungi in the MIN treatments was indicated by significantly increased ratios of ergosterol-to-microbial biomass C and the microbial biomass C/S ratio. Our results showed that complex interactions between the effects of fertilizer treatments and natural heterogeneity of soil pH existed for the majority of microbial biomass and activity indices.

Ergosterol and microbial biomass in the rhizosphere of grassland soils

Fungal and microbial biomass were determined by ergosterol and fumigation–extraction, respectively, in bulk grassland soil (<2 mm), rhizosphere and root material. The aim was to quantify the contribution of these three microbial fractions to the total soil microbial biomass and to soil organic matter. In the bulk soil, the average concentration was 3.37 μg g−1 for ergosterol, 860 μg g−1 for microbial biomass C, and 30.4 mg g−1 for organic C. In the rhizosphere material, the corresponding concentrations exceeded those of the bulk soil by 80, 80 and 50%, respectively. The large average ergosterol concentration of 74.2 mg g−1 root revealed a strong fungal colonisation of the root material. About 75% of the total ergosterol was found in the bulk soil fraction, 11% in the rhizosphere and 14% in the root material. In one soil nearly half of total ergosterol was found in the root material. However, the average ergosterol-to-biomass ratio in the root material was less than a third of the bulk soil or the rhizosphere soil, indicating that approximately two-thirds of CHCl3-labile C are presumably root-derived.