Limitations when quantifying microbial carbon and nitrogen by fumigation‐extraction in rooted soils

Soil microbial C and N (C_mic, N_mic) estimation by the chloroform fumigation‐extraction method is erroneous in densely rooted soils due to CHCl₃‐labile C and N compounds. The effect of a pre‐extraction with 50 mM K₂SO₄ and a pre‐incubation (conditioning at 25 °C for 7 days) on the flush in extractable, CHCl₃‐labile C (C‐flush) and N (N‐flush) was tested with reference to rooting density (0.3—75 mg root dry matter g^—1) in one arable and 3 grassland soils. In the arable soil and in the second horizon (10—20 cm) of a grassland soil, C‐flush values were not affected by the pre‐extraction. However, the pre‐extraction considerably reduced C‐flush values in the top soils of the grassland (above 10 cm). Only about 42 % was found in the pre‐extracted roots and the rest was lost during the pre‐extraction. The estimated concentrations of N_mic decreased due to pre‐extraction of soil samples with low root biomass. Clearly, the concentrations of N_mic were underestimated by introducing the pre‐extraction. Soil pre‐incubation reduced C‐flush values only slightly, whereas N‐flush values were not affected. It can be concluded that (1) CHCl₃‐labile root C and N is partly extracted with K₂SO₄ after pre‐incubation and (2) CHCl₃‐labile C and N removed with the roots during pre‐extraction is partly derived from microbial biomass. Soils with low rooting density (arable soils, grassland soils below approximately 10 cm depth) should therefore be fumigated and extracted without pre‐extraction. In densely rooted soils, fumigation extraction with and without pre‐extraction probably gives estimates for the minimum and maximum of C_mic and N_mic.     

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.     

Microbial biomass estimations in soils from tussock grasslands by three biochemical procedures

Microbial biomass was determined by three biochemical procedures in nine topsoils from a climosequence in tussock grasslands. The pH values of the samples ranged from 4.4 to 6.2 and organic C contents from 2.5 to 20.0%. When determined by a chloroform-fumigation procedure, contents of biomass C and mineral-N (Min-N) flush ranged from 530–2780 and 59–167 μgg^?1 dry soil respectively. Adenosine 5'-triphosphate (ATP) content ranged from 2.2 to 10.7 μg g^?1 dry soil. All three estimates were significantly correlated with each other and with several soil properties, including organic C and total N contents and CO₂ production. They were not significantly correlated with any climatic factor.In spite of these significant correlations, the ratios of the biomass estimates varied appreciably in the different soils. The ratios of biomass C/Min-N flush ranged from 7.8 to 22.8 (average 12.5), biomass C/ATP from 163 to 423 (average 248) and Min-N flush/ATP from 12 to 35 (average 22). These ratios were mostly higher than those found elsewhere for Australian and English soils. The high biomass C/ATP and Min-N flush/ATP ratios did not appear to originate from inefficient extraction of “native” ATP or from the soils' P status. Based on these results, care in the use of factors for obtaining soil microbial biomass content from Min-N flush or ATP values is indicated.     

Calibration model of microbial biomass carbon and nitrogen concentrations in soils using ultraviolet absorbance and soil organic matter

There is a need for a rapid, simple and reliable method of determining soil microbial biomass (SMB) for all soils because traditional methods are laborious. Earlier studies have reported that SMB‐C and ‐N concentrations in grassland and arable soils can be estimated by measurement of UV absorbance in soil extracts. However, these previous studies focused on soils with small soil organic matter (SOM) contents, and there was no consideration of SOM content as a covariate to improve the estimation. In this study, using tropical and temperate forest soils with a wide range of total C (5–204 mg C g^?1 soil) and N (1–12 mg N g^?1 soil) contents and pH values (4.1–5.9), it was found that increase in UV absorbance of soil extracts at 280 nm (UV₂₈₀) after fumigation could account for 92–96% of the variance in estimates of the SMB‐C and ‐N concentrations measured by chloroform fumigation and extraction (P < 0.001). The data were combined with those of earlier workers to calibrate UV‐based regression models for all the soils, by taking into account their varying SOM content. The validation analysis of the calibration models indicated that the SMB‐C and ‐N concentrations in the 0–5 cm forest soils simulated by using the increase in UV₂₈₀ and SOM could account for 86–93% of the variance in concentrations determined by chloroform fumigation and extraction (P < 0.001). The slope values of linear regression equations between measured and simulated values were 0.94 ± 0.03 and 0.94 ± 0.04, respectively, for the SMB‐C and ‐N. However, simulation using the regression equations obtained by using only the data for forest profile soils gave less good agreement with measured values. Hence, the calibration models obtained by using the increase in UV₂₈₀ and SOM can give a rapid, simple and reliable method of determining SMB for all soils.     

Microbial biomass and activity in soils amended with glucose

Four contrasting soils were amended with glucose at concentrations up to 10 mg g^?1 soil. The soils were incubated at 22°C for 14 days and the biomass determined at various times by chloroform fumigation or substrate-induced respiration. The adenosine triphosphate (ATP) content or the amylase and dehydrogenase activities were also determined. The size of the increases in biomass, ATP content and the enzyme activities was generally related to the amount of glucose added. The initially higher ATP levels quickly declined, and apparent substrate conversion figures up to 84% indicated that substrate-induced respiration overestimated the biomass. There were generally no significant correlations between ATP, biomass or enzyme activities.     

The effects of biocidal treatments on metabolism in soil—II. Gamma irradiation,autoclaving, air-drying and fumigation

Respiration and mineralisation of N were measured in a set of contrasting soils that had either been autoclaved, air-dried, fumigated (with chloroform or methyl bromide) or exposed to gamma radiation. The soils used were a manured and an unmanured arable soil, an acid and a neutral woodland soil, an arable sandy soil and an organic soil under grass. With the exception of the acid woodland soil, the flushes of decomposition (i.e. the increases in O₂ consumption, CO₂ evolution and N mineralisation that occurred when the treated soil was inoculated and incubated for 10 days) were in the order: air-drying < CH₃Br ? CHCl₃ < irradiation < autoclaving. All of the treatments, except air-drying, decreased the ratio (C mineralised after treatmcnt)/(N mineralised after treatment). All of the treatments increased the amount of 1N K₂SO₄ extractable organic C, autoclaving causing by far the greatest increase.Neither of the fumigants increased respiration in the acid soil over the whole 10 day period, although N mineralisation was slightly increased. Irradiation, air-drying and autoclaving did, however, produce a flush in the acid soil, the order being: irradiation < air-drying < autoclaving. A soluble substrate, extracted from yeast cells by ultrasonic disintegration, decomposed to about the same extent in neutral and in acid soil. When ¹⁴C labelled glucose was added to the acid soil and incubated for 52 days, the retention of labelled C was slightly greater (31·6%) than in a comparable near-neutral soil (28·8%). However, the flush that followed fumigation of the acid soil was only half that in the near-neutral soil, suggesting that less biomass is formed under acid conditions. Liming increased the size of the flush in an acid soil.For soils from the same field but under different management, the size of the flush caused by CHCl₃ is in the order: grassland > cropped arable > bare fallow. The flush is much more sensitive to differences in soil management than is the total amount of soil organic matter; a fallowed soil lost half its organic C in 10 yr whereas the increase in respiration that followed fumigation fell to one-seventh its original value. Two Nigerian soils behaved similarly; a soil that had been 2 years under cultivation contained only 16% less total organic C than an adjacent soil still under secondary forest, yet the flush in the cultivated soil was half that in the forest soil. The amount of substrate metabolised during the flush is thus very sensitive to changes in soil management that alter the amount of fresh organic matter entering the soil each year.     

Essai de correlation entre proprietes biochimiques d'un sol salsodique et sa biomasse

In order to study the influence of salinity on the biological activity of soils, experiments were performed in a saline alkaline soil from Tunisia (mediterranean semi-arid climate) and compared with results of similar experiments performed in a pelosol from a semi-continental climate (Lorraine, France). Both soils had received ¹⁴C-labelled maize straw.The microbial biomass was estimated by a modified Jenkinson's method and also by measurements of ATP content. The microbial activity was determined by measurements of total and ¹⁴CO₂ evolved.The results have shown an inverse correlation between the salinity determined by electrical conductivity and the biomass estimations and its activity. The higher ATP con tent (141.5 ng · 100 g^?1 soil) was observed in the pelosol and the lower (99.4 ng · 100 g^?1) in the saline soil. Simultaneously and respectively in the pelosol and the saline soil the biological carbon evolved was 114 and 54 mgC 100 g^?1 soil and the average rate of ¹⁴C mineralization was 0.82 and 0.45 mgC · 100 g^?1 soil.Results have also shown that CO₂ evolved after sterilization and reinoculation is not only provided by mineralization of microbial biomass during fumigation but also from interaction between organic-matter and CHCl₃; this interaction is more intense in the saline soil.     

Responses of carbon,nitrogen and phosphorus to two consecutive drying–rewetting cycles in soils

Drying and rewetting cycles are known to be important for the dynamics of carbon (C), phosphorus (P), and nitrogen (N) in soils. This study reports the short‐term responses of these nutrients to consecutive drying and rewetting cycles and how varying soil moisture content affects microbial biomass C and P (MBC and MBP), as well as associated carbon dioxide (CO₂) and nitrous oxide (N₂O) emissions. The soil was incubated for 14 d during which two successive drying–rewetting episodes were imposed on the soils. Soils subjected to drying (DRW) were rewetted on the seventh day of each drying period to return them to 60% water holding capacity, whilst continually moist samples (M), with soil maintained at 60% water holding capacity, were used as control samples. During the first seven days, the DRW samples showed significant increases in extractable ammonium, total oxidized nitrogen, and bicarbonate extractable P concentrations. Rewetting after the first drying event produced significant increases only in CO₂ flux (55.4 µg C g^?1 d^?1). The MBC and MBP concentrations fluctuated throughout the incubation in both treatments and only the second drying–rewetting event resulted in a significantly MBC decrease (416.2 and 366.8 mg kg^?1 in M and DRW soils, respectively). The two drying–rewetting events impacted the microbial biomass, but distinguishing the different impacts of microbial versus physical impacts of the perturbation is difficult. However, this study, having a combined approach (C, N, and P), indicates the importance of understanding how soils will react to changing patterns of drying–rewetting under future climate change.     

Effect of freeze-thaw events on mineralization of soil nitrogen

Summary In humid regions of the United States there is considerable interest in the use of late spring (April–June) soil NO ₃ ^– concentrations to estimate fertilizer N requirements. However, little information is available on the environmental factors that influence soil NO ₃ ^– concentrations in late winter/early spring. The influence of freeze-thaw treatments on N mineralization was studied on several central Iowa soils. The soils were subjected to temperatures of-20°C or 5°C for 1 week followed by 0–20 days of incubation at various temperatures. The release of soluble ninhydrin-reactive N, the N mineralization rate, and net N mineralization (mineral N flush) were observed. The freeze-thaw treatment resulted in a significant increase in the N mineralization rate and mineral N flush. The N mineralization rate in the freeze-thaw treated soils remained higher than in non-frozen soils for 3–6 days when thawed soils were incubated at 25°C and for up to 20 days in thawed soils incubated at 5°C. The freeze-thaw treatments resulted in a significant release of ninhydrin-reactive N. These values were closely correlated with the mineral N flush (r ²=0.84). The release of ninhydrin-reactive N was more closely correlated with biomass N (r ²=0.80) than total N (r ²=0.65). Our results suggest that freeze-thaw events in soil disrupt microbial tissues in a similar way to drying and re-wetting or chloroform fumigation. Thus the level of mineral N released was directly related to the soil microbial biomass. We conclude that net N mineralization following a spring thaw may provide a significant portion of the total NO ₃ ^– present in the soil profile.     

Decomposition of Zn-rich Arabidopsis halleri Litter in Low and High Metal Soil in the Presence and Absence of EDTA

Hyperaccumulating plants are increasingly investigated in combination with EDTA addition to soil for phytoremediation of heavy metal contaminated soils. A 60-day incubation experiment was carried out to investigate the effects of heavy metal release during the decomposition of Zn-rich (15.7 mg g^?1 dry weight) Arabidopsis halleri litter on C mineralization, microbial biomass C, biomass N, ATP, and adenylate energy charge (AEC). These effects were investigated in two soils with different Zn, Cu, and Pb levels, with and without EDTA addition to soil. The sole addition of Zn-rich A. halleri litter to the two soils did not increase the contents of NH₄NO₃ extractable Zn, only with the combined additions of EDTA and litter was there a considerable increase, being equivalent to three times the added amount in the low metal soil and to 50% in the high metal soil. Litter amendment increased the CO₂ evolved; being equivalent to 44% of the added C in the two soils, but EDTA addition had no significant effect on CO₂ evolution. Litter amendment resulted also in an 18% increase in microbial biomass C, 27% increase in ATP and 6% increase in AEC in the two soils, but EDTA had again no effect on these indices at both metal levels. In contrast, the sole addition of litter had no effect on microbial biomass N, but EDTA addition increased microbial biomass N on average by 49%. The application of EDTA for chelate-assisted phytoextraction should in the future consider the risk of groundwater pollution, which is intensified by resistance of EDTA to microbial decomposition.     

Microwave irradiation of soil for routine measurement of microbial biomass carbon

 Microwave irradiation was evaluated as a non-toxic alternate to chloroform fumigation for routine measurement of soil microbial biomass C. Microwave energy was applied to moist soil to disrupt microbial cells. The flush of C released was then measured after extraction or incubation. Microwave irradiation at 800 J g^–1 soil was optimal because this level resulted in an almost instantaneous rise in soil temperature (≥80  °C), an abrupt reduction in microbial activity, maximal release of biomass C, and minimal solubilization of humic substances. Both incubation-CO₂ titration and extraction-colorimetry methods were used on separate 20-g subsamples to compare the labile C in the microwave-treated and untreated soil samples. The incubation-titration method was also used to measure C in chloroform-fumigated soil samples. Averaged across soils, the chloroform fumigation yielded 123.3±5.1 mg CO₂-C kg^–1. Microwave irradiation yielded 93.6±3.9 mg CO₂-C kg^–1 soil determined by incubation and 52.4±2.4 mg C kg^–1 soil determined by extraction, accounting for 76% and 42% of the net flush of C measured by the chloroform fumigation. Microwave-stimulated net flushes of C were correlated closely (r ²=0.974 for incubation or 0.908 for extraction) with microbial biomass C measured by the chloroform fumigation. Little correlation was found with the total soil organic C (r ²=0.241 for incubation or for 0.166 extraction). Mean efficiency factors for incubation (K _MI) or extraction (K _ME) were used to calculate microbial biomass C from net flushes of C between microwaved and unmicrowaved soils. Values of K _MI and K _ME were not affected by soil pH, bulk density or clay contents. Extraction of microwaved soil by 0.5M K₂SO₄ proved to be a simple, fast, precise, reliable, and safe method to measure soil microbial biomass C. Received: 12 September 1997     

The proportional mineralisation of microbial biomass and organic matter caused by air-drying and rewetting of a grassland soil

During the first few days after rewetting of an air-dried soil (AD-RW), microbial activity increases compared to that in the original moist soil, causing increased mineralisation (a flush) of soil organic carbon (C) and other nutrients. The AD-RW flush is believed to be derived from the enhanced mineralisation of both non-biomass soil organic matter (due to its physical release and enhanced availability) and microbial biomass killed during drying and rewetting. Our aim was to determine the effects of AD-RW on the mineralisation of soil organic matter and microbial biomass during and after repeated AD-RW cycles and to quantify their proportions in the CO₂-C flushes that resulted. To do this, a UK grassland soil was amended with ¹⁴C-labelled glucose to label the biomass and then given five AD-RW cycles, each followed by 7 d incubation at 25 °C and 50% water holding capacity. Each AD-RW cycle increased the amount of CO₂-C evolved (varying from 83 to 240 μg g⁻¹ soil), compared to the control with, overall, less CO₂-C being evolved as the number of AD-RW cycles increased. In the first cycle, the amount of biomass C decreased by 44% and microbial ATP by 70% while concentrations of extractable C nearly doubled. However, all rapidly recovered and within 1.3 d after rewetting, biomass C was 87% and ATP was 78% of the initial concentrations measured prior to air-drying. Similarly, by 2 d, extractable organic C had decreased to a similar concentration to the original. After the five AD-RW cycles, the amounts of total and ¹⁴C-labelled biomass C remaining in the soil accounted for 60 and 40% of those in the similarly incubated control soil, respectively. Soil biomass ATP concentrations following the first AD-RW cycle remained remarkably constant (ranging from about 10 to 14 μmol ATP g⁻¹ biomass C) and very similar to the concentration in the fresh soil prior to air-drying. We developed a simple mathematical procedure to estimate the proportion of CO₂-C derived from biomass C and non-biomass C during AD-RW. From it, we estimate that, over the five AD-RW cycles, about 60% of the CO₂-C evolved came from mineralisation of non-biomass organic C and the remainder from the biomass C itself.     

Mineralization and immobilization of nitrogen in fumigated soil and the measurement of microbial biomass nitrogen

Immobilization of N was measured in a fumigated and in an unfumigated soil by adding (¹⁵NH₄)₂SO₄ and following the disappearance of inorganic label from the soil solution and its simultaneous conversion to soil organic N. Calculations based on the measurement of organically-bound ¹⁵N gave more consistent values for immobilization than did calculations based on the measurement of the disappearance of label from solution. The fumigated soil immobilized 6.6 μg N g^?1 N g^?1 soil in 10 days at 25°C, the unfumigated control 4.8 μg. The corresponding gross mineralization rates were 34.9 and 5.6 μg N g^?1 soil in 10 days.Addition of 58 μg N as (¹⁵NH₄)₂SO₄ to the fumigated soil increased the quantity of the ynlabelled NH₄-N extracted at the end of 10 days from 33.8 to 37.8 μg Ng^?1 soil, i.e. there was a positive Added Nitrogen Interaction (ANI). The added labelled N produced this ANI, not by increasing the rate of mineralization of organic N, but by standing proxy for unlabelled N that otherwise would have been immobilized.A procedure for calculating biomass N from the size of the flush of mineral N caused by fumigation is proposed. Biomass N (B_N) is calculated from the relationship B_N = F'_N/0.68 where F'_N is [(N in fumigated soil incubated for 10 days — (N in unfumigated soil incubated for 10 days)].     

Estimation of the adenylate energy charge in unamended and amended agricultural soils

To determine relatively low concentrations of adenine nucleotides in agricultural soils a NaHCO₃-based extradant was developed and compared with the trichloroacetic acid-paraquat-phosphate extradant. The new medium, consisting of chloroform, sodium bicarbonate, phosphate and adenosine (pH 8.0) gave soil extracts which could be investigated without further neutralization and dilution. ATP was measured directly in the soil extracts by the luciferin-luciferase system. ADP and AMP were estimated after their enzymatic conversion to ATP by standard methods. The quantities of nucleotides corrected for recovery of standards were used to calculate the adenylate energy charge (AEC) from the formula AEC = [ATP] + 1/2[ADP]/[ATP] + [ADP] + [AMP], The AEC was estimated in six unplanted soils from agricultural fields. A very similar energy charge of 0.3-0.4 was found in all soils sampled which indicates a low metabolic activity of the soil population. Two other soils with a pronounced difference in biomass-C content were used to investigate the influence of different amendments on the AEC. In an experiment with low glucose supplements up to 500 μg C g^?1 soil, the soil with the low biomass-C (a cambisol) showed a distinct increase of the AEC from 0.34 to 0.50, whereas the soil with the high biomass-C content (a phaeozem) increased its AEC only slightly from 0.32 to 0.37. In another experiment with high glucose supplements the phaeozem reached its maximum AEC value of 0.56 after the addition of 4000 μg Cg^?1 soil. An amendment with 8000 μg C g^?1 soil gave no further increase. In the combisol the addition of 1000 μg C g^?1 soil increased the AEC to 0.61. Higher supplements gave only a slight further increase to a maximum value of 0.67 after the addition of 8000 μg C g^?1 soil. The same AEC value was reached when the cambisol was amended with a mixture of organic substrates at a concentration of 10,000 μg C g^?1 soil.     

The effects of biocidal treatments on metabolism in soil—I. Fumigation with chloroform

This series of five papers is a study of how biocidal treatments influence metabolism in soil, directed particularly towards the flush of decomposition caused by fumigation, and designed to see if the size of this flush can be used as a measure of the soil biomass.Chloroform fumigation caused an immediate increase in the amounts of ammonium and organic C extracted from a soil by 1 N K₂SO₄. When the CHCl₃-treated soil was then inoculated with fresh soil and incubated for 10 days. it consumed 2·8 times more O₂, evolved 2·2 times more CO₂ and mineralised 7·3 times more N than an unfumigated soil. Extractable organic C decreased by about 40% when the fumigated soil was incubated for 10 days. A second fumigation given immediately after the first produced no further increase in the flush, but some recovery occurred if the soil was incubated between fumigations. However, this recovery was slow and incomplete; a second fumigation given 53 days after the first gave a flush only one-seventh the size of the first. Glucose (or ryegrass) added to the soil and allowed to decompose before fumigation increased the size of the flush. After a 52-day incubation, 29% of the C originally added as ¹⁴C labelled glucose remained in the soil; fumigation on the 52nd day increased the evolution of labelled CO₂ during the subsequent 10-day period by a factor of 8. Fumigation of a soil that had already been sterilized by 2·5 Mrads of gamma radiation increased the flush slightly; the amount of O₂ consumed in 10 days increased from 123 to 137 mg/100 g soil. It is proposed that the flush of decomposition following CHCl₃ fumigation is caused by the decomposition of killed organisms by the survivors (or by organisms added in the inoculum) and that organisms are more rapidly and completely attacked after exposure to CHCl₃ than after irradiation. On this hypothesis. 10% of the glucose C originally added to the soil was located in the soil biomass after 52 days.     

Laboratory Assessment of Nostoc 9v (Cyanobacteria) Effects on N2 Fixation and Chemical Fertility of Degraded African Soils

The potential of Nostoc 9v for improving the nitrogen (N)₂–fixing capacity and nutrient status of semi‐arid soils from Tanzania, Zimbabwe, and South Africa was studied in a laboratory experiment. Nostoc 9v was inoculated on nonsterilized and sterilized soils. Inoculum rates were 2.5 mg dry biomass g^?1 soil and 5 mg dry biomass g^?1 soil. The soils were incubated for 3 months at 27 °C under 22 W m² illumination with a photoperiod of 16 h light and 8 h dark. The moisture was maintained at 60% of field capacity. In all soils, Nostoc 9v proliferated and colonized the soil surfaces very quickly and was tolerant to acidity and low nutrient availability. Cyanobacteria promoted soil N₂ fixation and had a pronounced effect on total soil organic carbon (SOC), which increased by 30–100%. Total N also increased, but the enrichment was, in most soils, comparatively lower than for carbon (C). Nitrate and ammonium concentrations, in contrast, decreased in all the soils studied. Increases in the concentration of available macronutrients were produced in most soils and treatments, ranging from 3 to 20 mg phosphorus (P) kg^?1 soil, from 5 to 58 mg potassium (K) kg^?1 soil, from 4 to 285 mg calcium (Ca) kg^?1, and from 12 to 90 mg magnesium (Mg) kg^?1 soil. Positive effects on the levels of available manganese (Mn) and zinc (Zn) were also observed.     

A simple method for quantitative determination of ATP in soil

A simple method to measure soil ATP by the luciferin-luciferase system is described. The ATP is extracted from the soil by vigorous shaking with a sulfuric acid-phosphate solution for 15 min. An aliquot of the soil suspension is neutralized with a Tris-EDTA solution and mixed with a special ATP releasing reagent (NRB). ATP is measured after a 10 s exposure to the NRB reagent, followed by addition of luciferin-luciferase and integration over 10 s in a Lumacounter M 2080. The ATP content in soils which had been stored at 5°C for 90 days and then incubated at 25°C for 5 days, ranged from 0.37 to 7.52 μg ATP g^?1 dry wt, with standard deviations less than 10%. There was a close (r = 0.96) linear relationship between ATP content and biomass C determinated by fumigation for this group of soils. The soil biomass contained 4.2–7.1 μg ATP mg^?1 biomass C. The ATP content of the biomass declined during storage at 5°C for 210 days.     

Correlation between four methods to estimate total microbial biomass in stored,air-dried and glucose-amended soils

Correlation between the microbial volume, chloroform fumigation (CO₂-C flush), substrateinduced respiration (SIR) and ATP content methods to estimate microbial biomass was assessed on three New Zealand soils (two grassland, one arable) under three different treatments (stored, air-dried and glucose-amended). There were significant, positive correlations between all methods, r = 0.69–0.88, which were improved, r = 0.71–0.96, if the data for air-dried or glucose-amended soils were excluded from the analyses. The best agreement was between CO₂-C flush and ATP and the worst between CO₂-C flush and microbial volume. Exclusion of air-dried soil data improved these correlations.Estimates of microbial biomass for each soil often differed significantly between the four methods, when conversion factors cited in the literature were used. Ratios (i.e. conversion factors) between CO₂-C flush and ATP or SIR, or SIR and volume, were different to those cited in the literature, and only similar if specific data were excluded.We recommend that a minimum of two and preferably three methods be used to quantify the microbial population of soil, and that emphasis should be placed on the relative differences within and between soils using data which have not been converted to biomass C. Conversion of data to biomass C may result in substantial errors.     

Microbial biomass and respiration in soils derived from lignite ashes: a profile study

Microbial biomass C and soil respiration measurements were made in 17–20 yr old soils developed on sluiced and tipped coal‐combustion ashes. Topsoil (0–30 cm) and subsoil (30–100 cm) samples were collected from three soil profiles at two abandoned disposal sites located in the city area of Halle, Saxony‐Anhalt. Selected soil physical (bulk density and texture) and chemical (pH, organic C, total N, CEC, plant available K and P, and total Cd and Cu) properties were measured. pH values were significantly lower while organic C and total N contents and the C : N ratio were significantly higher in the topsoil than in the subsoil indicating the effects of substrate weathering and pedogenic C accumulation. Likewise, microbial biomass C, K₂SO₄‐extractable C, and soil respiration with median values of 786 μg biomass C g^–1, 262 μg K₂SO₄‐C g^–1, and 6.05 μg CO₂‐C g^–1 h^–1, respectively, were significantly higher in the topsoil than in the subsoil. However, no significant difference was observed in metabolic quotient between the topsoil and the subsoil. Metabolic quotient with median values of 5.98 and 8.54 mg CO₂‐C (g biomass C)^–1 h^–1 for the 0–30 cm and 30–100 cm depths, respectively, was higher than the data reported in the literature for arable and forest soils. Microbial biomass C correlated significantly with extractable C but no relationship was observed between it and total N, Cd, and Cu contents, as well as plant‐available K and P. We conclude that the presence of the remarkable concentration of extractable C in the weathered lignite ashes allowed the establishment of microbial populations with high biomass. The high metabolic quotients observed might be attributed to the heavy‐metal contamination and to the microbial communities specific to ash soils.     

Heat output of the soil biomass

Amending soils with glucose (5 mg g^?1) resulted in an immediate increase in microbial activity and within 30 min the rates of heat output and respiration at 22° C were increased by up to 17.8 and 23.4 times, respectively. The increased rate of heat output remained stable for up to 6 h and there was good correlation with the amount of CO₂ respired. The soil biomass was calculated by the method of Anderson and Domsch (1978). The rate of heat output of the biomass varied in different soils and ranged from 11.5 to 83.7 Jh^?1 g^?1 biomass C. In glucose-amended soils, however, the rate of heat output was much more consistent; the soils were in two groups having between 169–265 Jh^?1g^?1 biomass C or 454–482 J h^?1 g^?1 biomass C, both the latter two soils were from pasture. The increased rate of heat output from the amended soils was lower than expected from the respiration rate and the heat of oxidation of glucose, suggesting that a proportion of the CO₂ respired was from catabolism of substrates other than glucose. Use of ¹⁴C-glucose confirmed that between 57–91% of the CO₂ was derived from the glucose substrate.