Adenosine triphosphate and microbial biomass in soil

Two methods for measuring adenosine 5'-triphosphate (ATP) in soil were compared, one based on extraction with NaHCO₃-CHCl₃ and thel other on extraction by a trichloracetic acid-phosphate-paraquat reagent. Recoveries of added ATP were greater with the NaHCO₃-CHCl₃ reagent but the extraction of “native” soil ATP by NaHCO₃-CHCl₃ was only about a third of that by TCA-phosphate-paraquat.Microbial biomass C and ATP were measured in 8 contrasting English soils, using the fumigation method to measure biomass C and the TCA-phosphate-paraquat method to measure ATP. Except in one acid woodland soil, the ratio (ATP content of the soil)/(biomass C content of the soil) was relatively constant, with a mean of 7.3 mg ATP g^?1 biomass C for the different soils. This value is very similar to that obtained earlier in a range of 11 grassland and arable soils from Australia. Taking the English and Australian grassland and arable soils together, there is a close (r = 0.975) linear relationship between ATP and microbial biomass C that holds over a wide range of soils and climates. From this relationship, the soil biomass contains 7.25 mg ATP g^?1 biomass C, equivalent to an ATP-to-C ratio of 138, or to 6.04 μmoles ATP g^?1 dry biomass.The acid woodland soil (pH 3.9) contained much less biomass C, as measured by the fumigation method, than would have been expected from this relationship. This, and other evidence, suggests that the fumigation method for measuring microbial biomass C breaks down in strongly acid soils.The ATP content of the biomass did not depend on the P status of the soil, as indicated by NaHCO₃-extractable P.     

Adenylates in the soil microbial biomass at different temperatures

Five soils from temperate sites (Germany; 2 arable and 3 grassland) were incubated aerobically at 5, 10, 15, 20, 25, 35, and 40 °C for 8 days. Soils were analysed for soil microbial biomass C, biomass N, AMP, ADP, and ATP to determine whether the increase in the ATP-to-microbial biomass C ratio with increasing temperature was either due to an increase in the adenylate energy charge (AEC) or de novo synthesis of ATP, or both. Around 80% of the variance in microbial biomass C and biomass N was explained by differences in soil properties, only 7% by the temperature treatments. Averaging the data of all 5 soils for each incubation temperature, the microbial biomass C content decreased with increasing temperature from 15 to 40 °C continuously by 2.5 μg g⁻¹ soil °C⁻¹ after 8-days' incubation. However, this decrease was not accompanied by a similar decrease in microbial biomass N. The average microbial biomass C/N ratio was 6.8. Between 54 and 76% of the variance in AMP, ADP, ATP and the sum of adenylates was explained by differences in soil properties and between 14 (ADP) and 27% (ATP) by the temperature treatments. However, temperature effects on AMP and ADP were variable and inconsistent. In contrast, ATP and consequently also the sum of adenylates increased continuously from 5 to 30 °C followed by a decline to 40 °C. The AEC showed similarly a small, but significant increase with increasing temperature from 0.73 to 0.85 at 30 °C. Consequently, the majority of the variance, i.e. roughly 60% in AEC values, but also in ATP-to-microbial biomass C ratios was explained by the incubation temperature. The mean ATP-to-microbial biomass C ratio increased from 4.7 μmol g⁻¹ at 5 °C to a 2.5 fold maximum of 12.0 μmol g⁻¹ at 35 °C. This increase was linear with a rate of 0.26 μmol ATP g⁻¹ microbial biomass C °C⁻¹. The energy for the extra ATP produced during temperature increase is probably derived from an accelerated turnover of endocellular C reserves in the microbial biomass.     

Ecophysiology of the soil microbial biomass and its relation to the soil microbial N pool

Abstract. Microbial osmoregulation as a factor regulating the nitrogen and carbon contents of soil microbial biomass was studied in two experiments. In the first the percentages of the carbon and nitrogen occurring in the cytoplasm of Aspergillus flavus and Pseudomonas sp. were shown to be strongly influenced by osmotic stress. In the second, biomass carbon and nitrogen initially increased with increasing water stress (osmotic and matric) up to −1.0 and −1.5 MPa, respectively, but declined under greater osmotic stress. As the soil microbial carbon and nitrogen pools are affected by these stresses, allowance must be made for them when interpreting biomass measurements in water-stressed soils.     

Ratios of microbial biomass estimates to evaluate microbial physiology in soil

Soil microbial biomass data derived from fumigation–extraction (FE), substrate-induced respiration (SIR) and ATP estimations differed significantly and were significantly correlated, which agrees to previous studies. In a second step, the SIR/FE, ATP/FE and SIR/ATP ratios were calculated to evaluate the glucose-responsive and active component of the microbial (active and resting) biomass and the glucose-responsive component of the active microbiota. Soils were sampled along gradients within and between associated ecosystems in Northern Germany, Denmark and along a gradient of heavy metal pollution in Finland. The ratios indicated that the active portion and glucose-responsive component decreased with proceeding litter decomposition, higher degree of sustainable land management practices and higher degree of heavy metal contamination. This work was presented at the workshop ‘Non-molecular manipulation of soil microbial communities’ at the University of Udine, Udine, Italy, 17–20 October 2004; convened by P.C. Brookes and M. De Nobili and supported by European Science Foundation.     

The significance of microbial biomass sulphur in soil

The soil microbial biomass S fraction of total organic S in soil is considered to be relatively labile and the most active S pool for S turnover in soil. Its significance has been demonstrated in studies of S deficiency in agronomic situations and in those of S pollution from high atmospheric inputs. The utility of the CHCl₃ fumigation-extraction technique for the measurement of microbial S has been proved for a range of soils and conditions. The various methodologies currently available are discussed, including the need for determination of the conversion (K _s) factor. Microbial S values, summarized from the available literature, ranged from 3 to 300 g S g^-1 dry weight soil. They were generally greater in grassland than in arable systems, though the greatest values were obtained in the few examples from forest and peatland soil systems. Microbial S values showed direct relationships with both microbial C and with total soil organic S. Again, there were significant differences between arable and grassland systems. The effect of factors such as organic and inorganic inputs as well as soil physical conditions on microbial S are described. Microbial S turnover rates were estimated from seasonal, ³⁵S-labelling and modelling studies. These rates varied between an approximately annual turnover rate in undisturbed soils up to 80 year^-1 following the addition of readily available substrates. Prospective future research areas are also outlined.     

Secondary salinity effects on soil microbial biomass

Secondary soil salinilization is a big problem in irrigated agriculture. We have studied the effects of irrigation-induced salinity on microbial biomass of soil under traditional cotton (Gossypium hirsutum L.) monoculture in Sayhunobod district of the Syr-Darya province of northwest Uzbekistan. Composite samples were randomly collected at 0–30 cm depth from weakly saline (2.3 ± 0.3 dS m⁻¹), moderately saline (5.6 ± 0.6 dS m⁻¹), and strongly saline (7.1 ± 0.6 dS m⁻¹) replicated fields, 2-mm sieved, and analyzed for pH, electrical conductivity, total C, organic C (C_Org), and extractable C, total N and P, and exchangeable ions (Ca²⁺, Mg²⁺, K⁺, Na⁺, Cl⁻, and CO₃²⁻), microbial biomass (C_mic). The Na⁺ and Cl⁻ concentrations were 36-80% higher in strongly saline compared to weakly saline soil. The C_Org concentration was decreased by 10% and C_Ext by 40% by increasing soil salinity, whereas decrease in C_mic ranged from 18-42% and the percentage of C_Org present as C_mic from 8% to 26%. We conclude that irrigation-induced secondary salinity significantly affects soil chemical properties and the size of soil microflora.     

Changes in the structure of soil microbial biomass under fallow

The number and biomass of various groups of microorganisms in fallow soils is greater as compared to plowed soils. The microbial biomass in all fallow and plowed soils is dominated by fungal mycelium (from 90% in the top horizons to 97% in the lower ones). The part of spores in the fungal biomass is higher in plowed soils (from 9% in the top horizons to 4% in the lower ones) as compared to fallow soils (3.5?C6%). The fallow soils are characterized by the greater part of prokaryotic microorganisms in the biomass, and the reserves and structure of the microbial biomass are more similar to those in the undisturbed soils. These characteristics changed during a ten-year-long period in a soddy-calcareous soil and during a 25-year-long period in a leached chernozem.     

Interactions between microclimatic variables and the soil microbial biomass

Summary Soil moisture, temperature, microbial substrate-induced respiration and basal respiration were monitored in two plots in an agricultural field from April 30 to September 25, 1987, and in a further two plots from May 26 to August 27, 1988. An attempt to relate biological variables to microclimatic variables was made through the use of correlation analysis. The microbial substrate-induced and basal respiration were both strongly positively correlated with the soil moisture content, and to a lesser extent positively related to soil temperature, especially when partial correlation was used to control for variation in soil moisture. Short-term changes in substrate-induced and basal respiration were correlated with changes in soil moisture but were largely independent of soil temperature. The ratio of basal to substrate-induced respiration (indicating the respiration: biomass ratio and therefore ecosystem stability or persistence) was negatively associated with the soil moisture content and in some instances with soil temperature when partial correlation analysis (correcting for soil moisture variation) was used. This suggests that the climatic conditions which contributed to the lowest ecosystem stability were low temperature, low moisture conditions.     

Measuring soil microbial biomass using an automated procedure

Here we outline the development of the first automated procedure for measuring soil microbial biomass carbon (biomass C) by Fumigation-Extraction (FE) and on which this Citation Classic is based. The method (and its later variations) has been used widely since it was published. It gives essentially the same results as the Fumigation-Incubation (FI) method on which it was based and which it has now largely replaced. Analysis of the current data clearly showed that the calibration value to convert extracted organic C to biomass C (k_EC = 0.45) using FE is still valid and that there was no need for a change. We also review some of the previous discussions about the method and outline future prospects for microbial biomass measurements in soil microbial ecology.     

C and N natural abundance of the soil microbial biomass

Stable isotope analysis is a powerful tool in the study of soil organic matter formation. It is often observed that more decomposed soil organic matter is ¹³C, and especially ¹⁵N-enriched relative to fresh litter and recent organic matter. We investigated whether this shift in isotope composition relates to the isotope composition of the microbial biomass, an important source for soil organic matter. We developed a new approach to determine the natural abundance C and N isotope composition of the microbial biomass across a broad range of soil types, vegetation, and climates. We found consistently that the soil microbial biomass was ¹⁵N-enriched relative to the total (3.2 ‰) and extractable N pools (3.7 ‰), and ¹³C-enriched relative to the extractable C pool (2.5 ‰). The microbial biomass was also ¹³C-enriched relative to total C for soils that exhibited a C3-plant signature (1.6 ‰), but ¹³C-depleted for soils with a C4 signature (−1.1 ‰). The latter was probably associated with an increase of annual C3 forbs in C4 grasslands after an extreme drought. These findings are in agreement with the proposed contribution of microbial products to the stabilized soil organic matter and may help explain the shift in isotope composition during soil organic matter formation.     

Effect of organic-complexed superphosphates on microbial biomass and microbial activity of soil

Organic complexed super-phosphates (CSPs) are formed by the complexation of humic acid (HA) with calcium monophosphate. The aim of this study was to determine whether two CSPs, characterized by different HA concentrations, added to a calcareous soil at an agronomic dose, were able to maintain the phosphorus (P) in a soluble form longer than the superphosphate fertilizer. Another important goal was to verify if CSP could positively influence soil microbial biomass and soil microbiological activities. Organic complexed super-phosphates were capable of keeping a large portion of P in a soluble form under different soil water conditions. In particular, the CSP with the highest organic C content was the most effective product, capable of maintaining, in an available form, the 73 % of the initially added P at the end of the experiment. In addition, it was the most effective in increasing C–CO₂ soil emission, microbial biomass carbon (C) and nitrogen (N), fluoresceine diacetate hydrolysis and activities of alkaline phosphomonoesterase, β-glucosidase and urease. The addition of CSPs to soil probably produced a priming effect, increasing several times C–CO₂ release by the treated soil. The significant correlation (p?<?0.05) between C–CO₂ emission and the amount of C added to soil by CSP suggests that the added HA acted as trigger molecules.     

Estimation of microbial biomass and activity in soil using microcalorimetry

Relationships between the rate of heat output from soil, the rate of respiration and the soil microbial biomass were investigated for 25 soils from northern Britain. The rate of heat output, measured in a Calvet microcalorimeter at 22°C, correlated well with the rate of carbon dioxide respiration. The average amount of heat evolved per cm³ of gas respired. 21.1 J cm^?3, suggests that the biomass metabolism was largely aerobic. The rate of heat output per unit of total microbial biomass was remarkably uniform over a wide range of soils, but showed differences depending upon whether the soil had been stored or amended. Mineral soils that had been stored at 4°C had the lowest heat output, 12.0 mW g^?1 biomass C, compared with a mean of 20.4 mW g^?1 biomass C for freshly-collected soils. Amendment with glucose (0.5% w/w) caused an immediate increase in respiration and heat output, up to 59.4 mW g^?1 biomass C for stored soils and 188.2 mW g^?1 biomass C for freshly collected soils. There was a consistent relationship between the biomass and the rate of heat output from freshly collected and amended mineral and organic soils which gave a linear fit using log transformed data: y= 0.6970+ 1.025x (r= 0.98, P < 0.001) (y=log₁₀ biomass C, μgC g^?1; x=log₁₀ rate of heat output at 22°C, μW g^?1). The overall relationship between biomass and the rate of heat output for all the amended samples was: 1 g biomass C= 180.05 ± 34.61 mW.     

Limitations of soil microbial biomass carbon as an indicator of soil pollution in the field

The size of the soil microbial biomass carbon (SMBC) has been proposed as a sensitive indicator for measuring the adverse effects of contaminants on the soil microbial community. In this study of Australian agricultural systems, we demonstrated that field variability of SMBC measured using the fumigation-extraction procedure limited its use as a robust ecotoxicological endpoint. The SMBC varied up to 4-fold across control samples collected from a single field site, due to small-scale spatial heterogeneity in the soil physicochemical environment. Power analysis revealed that large numbers of replicates (3-93) were required to identify 20% or 50% decreases in the size of the SMBC of contaminated soil samples relative to their uncontaminated control samples at the 0.05% level of statistical significance. We question the value of the routine measurement of SMBC as an ecotoxicological endpoint at the field scale, and suggest more robust and predictive microbiological indicators.     

Synthesis of 15N-labelled microbial biomass in soil in situ and extraction of biomass N

Summary A Pakistani soil (Hafizabad silt loam) was incubated at 30°C with varying levels of ¹⁵N-labelled ammonium sulphate and glucose (C/N ratio of 30 at each addition rate) in order to generate different insitu levels of ¹⁵N-labelled microbial biomass. At a stage when all of the applied ¹⁵N was in organic forms, as biomass and products, the soil samples were analysed for biomass N by the chloroform (CHCl₃) fumigation-extraction method, which involves exposure of the soil to CHCl₃ vapour for 24 h followed by extraction with 500 mM K₂SO₄. A correction is made for inorganic and organic N in 500 mM K₂SO₄ extracts of the unfumigated soil. Results obtained using this approach were compared with the amounts of immobilized ¹⁵N extracted by 500 mM K₂SO₄ containing different amounts of CHCl₃. The extraction time varied from 0.5 to 4 h.The amount of N extracted ranged from 27 to 270 g g^–1, the minimum occurring at the lowest (67 g g^–1) and the maximum at the highest (333 g g^–1) N-addition rate. Extractability of biomass ¹⁵N ranged from 25% at the lowest N-addition rate to 65%a for the highest rate and increased consistently with an increase in the amount of ¹⁵N and glucose added. The amounts of both soil N and immobilized ¹⁵N extracted with 500 mM K₂SO₄ containing CHCl₃ increased with an increase in extraction time and in concentration of CHCl₃. The chloroform fumigation-extraction method gives low estimates for biomass N because some of the organic N in K₂SO₄ extracts of unfumigated soil is derived from biomass.     

A microplate assay to measure soil microbial biomass phosphorus

Quantification of phosphorus (P) concentrations in microbial biomass is required to better understand how P immobilization and turnover in soils are controlled by environmental and anthropogenic factors. Soil microbial biomass P (MBP) is generally extracted using the chloroform fumigation-direct extraction procedure and then analysed for P using the ammonium molybdate-ascorbic acid method on a flow injection analysis (FIA) system. Our objective was to determine whether a microscale malachite green method on a microplate system would provide as accurate MBP analysis as the ascorbic acid method on an FIA system. Twelve soils were collected from agricultural fields in southwestern Quebec, fumigated with chloroform and extracted with 0.5 M NaHCO₃ (pH 8.5). The dissolved inorganic phosphorus (DIP) concentration in fumigated soils was not affected by the method of analysis, and results from the two systems of analysis were significantly correlated (r =0.998, P <0.05). The MBP concentrations in these agricultural soils were between 0.36 and 60.05 g P g^–1, consistent with other published values. Our results indicate that MBP can be assessed equally well with the malachite green method using a microplate system as with the ascorbic acid method on an FIA system. The microplate system is rapid and requires smaller volumes of samples and reagents than the FIA system, thus reducing the quantity of waste produced. We conclude that the microscale malachite green method could be applied to measure the MBP concentration in a wide range of soils with good sensitivity, reproducibility and accuracy.     

Effect of soil CO2 concentration on microbial biomass

The effect of increasing soil CO₂ concentration was studied in six different soils. The soils were incubated in ambient air (0.05 vol.% CO₂) or in air enriched with CO₂ (up to 5.0 vol.% CO₂). Carbon dioxide evolution, microbial biomass, growth or death rate quotients and glucose decay rate were measured at 6, 12 and 24 h of CO₂ exposure. The decrease in soil respiration ranged from 7% to 78% and was followed by a decrease in microbial biomass by 10–60% in most cases. High CO₂ treatments did not affect glucose decay rate but the portion of C_gluc mineralized to CO₂ was lowered and a larger portion of C_gluc remained in soils. This carbon was not utilized by soil microorganisms. Received: 30 August 1996     

Seasonal dynamics of soil microbial biomass in coastal sand dune forest

Sand dunes are a typical landscape in the coast of western Taiwan, where Casuarina forests were established decades ago to stabilize sand dunes and protect the inland vegetation. Study of microbial biomass in such an ecosystem may give insights into the role of microbes in soil fertility and nutrient cycling. We established our study sites in two topographic units based on elevation and drainage types: upland and lowland. The study lasted for 2 years, and soil samples were collected every 3 months. Microbial biomass C (C_mic) and N (N_mic) were high in a shallow humic layer that rested on top of the soil (1222–1319 mg kg⁻¹ for C_mic and 245–276 mg kg⁻¹ for N_mic) and declined sharply to only one-tenth of the above values in the underlying surface soil (0–10 cm depth). Microbial biomass C_mic and N_mic in humic and surface soil were not significantly different between upland and lowland sites. In the upland soils, the mean C_mic was highest in autumn for both the humic and surface soil, and lowest in spring and summer for the humic layer and summer for the surface soil layer. In the lowland soils, the C_mic was highest in winter for both humic and surface soil, and lowest in spring and autumn for the humic layer and spring and summer for surface soil. Strong fluctuations of C_mic and N_mic were associated with the soil moisture prior to sampling, which appeared to control the size of microbial biomass in this environment. Temperature had little effect on the dynamics of soil microbial biomass in the sand dune forest ecosystem.     

Spatial structure of microbial biomass and activity in prairie soil ecosystems

Management of soil ecosystems requires assessment of key soil physicochemical and microbial properties and the spatial scale over which they operate. The objectives were to determine the spatial structure of microbial biomass and activity and related soil properties, and to identify spatial relationships of these properties in prairie soils under different management histories. Soil were sampled along a transect at 0.2 m intervals in each of five long-term treatments, namely, undisturbed, cattle grazed at two intensities, and cultivated with either wheat (Triticum aestivum L.) or cotton (Gossypium hirsutum L.). Contents of organic carbon (C_org), dissolved organic C (DOC), soluble nitrogen (N_sol), and microbial biomass C (C_mic) and N (N_mic) as well as dehydrogenase activity (DH) in 70 samples were evaluated. Results showed that long-term soil management altered the spatial structure and dependence of C_org and microbial biomass and activity. Cultivation has contributed to high nugget variance for C_org, C_mic, N_mic and DH which interfered with detection of spatial structure at the sampling scale used. Contents of C_org were spatially connected to microbial biomass and activity and to DOC in the uncultivated but not in the cultivated soils, indicating that various factors affected by management may operate at different spatial scales.