Adenosine triphosphate (ATP) and microbial biomass in soil: Effects of storage at different temperatures and at different moisture levels

Abstract

On air‐drying, the ATP contents of two moist soils fell to about one quarter of their original values. When a freshly‐sampled soil (field temperature 5.5°C) was stored moist (43% water holding capacity) for 7 days at 25°C the ATP content increased from 4.54 to 7.84 μg ATP g^‐1 soil. Storage at 10°C caused a smaller increase; to 5.39 μg g^‐1 soil. Microbial biomass C also increased on storage but the relative increase was less than that of ATP. Thus the biomass C/ATP ratio fell from 234 in the freshly sampled soil to 168 in the soil stored moist for 7 days at 25°C. The ATP content declined to less than half its starting value if storage was under waterlogged conditions.

The ATP method for determining microbial biomass in soil depends on the use of a constant factor (5.85 mg ATP g^‐1 biomass C) for converting ATP content to biomass C. This factor came from work on soils that had been stored moist at 25°C for several days before biomass C and ATP measurements were made: it is only applicable to soils that have been stored in this way.     

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.     

Adenylate energy charge measurements in soil

Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphatc (ADP) and adenosine 5'-monophosphate (AMP) were extracted from soil with either a solution of trichloroacetic acid, paraquat and phosphate (TCA reagent) or a mixture of chloroform, sodium hydrogen carbonate, phosphate and adenosine (NaHCO₃ reagent). Standard enzymic procedures were used to convert ADP and AMP to ATP, which was measured by the fire-fly luciferin-luciferase system. The measured quantities of nucleotides were corrected for incomplete extraction using the percentage recoveries of added ATP, ADP and AMP. The adenylate energy charge ratio (AEC) was calculated from the formula AEC = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]).Measurements were made on a grassland soil, following a conditioning incubation at 15°C and 50% WHC for 7 days. Additional measurements were made on the same soil after a further 50- or 100-day incubation at 25°C and 50% WHC, with or without an amendment of 1100 μg ryegrass Cg⁻¹ soil, added at the end of the conditioning incubation. Biomass-ATP concentration, measured in TCA extracts, changed little, even on prolonged incubation, and was maintained at a level comparable to that observed in earlier work (about 10 p mol ATP g⁻¹ biomass C). AEC values in TCA soil extracts were high (0.8–0.9) for all soil treatments and independent of substrate addition or length of incubation.In contrast, AEC was low (0.4) in fresh soil extracted with NaHCO₃ reagent, but increased to 0.6 when ryegrass was incubated with the soil for 50 days. Although the total adenine nucleotide pool (i.e. [ATP] + [ADP] + [AMP]) was similar as measured in NaHCO₃ and in TCA soil extracts, both energy charge and ATP content were lower in the NaHCO₃ extracts. It was therefore concluded that the main reason for the lower AECs observed with the NaHCO₃ reagent was that microbial ATPases were still active during extraction and caused appreciable hydrolysis of microbial ATP to ADP and AMP. In contrast, the TCA reagent rapidly inactivates ATPases and is therefore preferable for extracting adenine nucleotides from soil.The results indicate that the soil microbial biomass, although a mainly dormant population, maintains both AEC and ATP at levels characteristic of exponentially growing organisms in vitro, even during prolonged incubation without fresh substrate. It was also concluded that roots make a negligible contribution to total ATP extracted from fresh sieved soil.     

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 adenylate energy charge of the soil microbial biomass

A method was developed for measuring adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) in soil. All three adenine nucleotides were extracted from soil with a solution of trichloroacetic acid, paraquat and phosphate. ATP was measured in the neutralised (pH 7.4) soil extracts by the fire-fly luciferin-luciferase system. ADP was measured as ATP after incubating the neutralised extracts with pyruvate kinase (PK) and phosphoenolpyruvate (PEP) to convert ADP to ATP. AMP was converted to ATP by incubation with the coupled PK-PEP-myokinase system and measured as ATP. The quantities of nucleotides present in the extracts were corrected for incomplete extraction from soil by measuring the percentage recovery of added ATP, ADP and AMP. The adenylate energy charge (AEC) was calculated from the formula AEC = [[ATP] + 0.5[ADP]]/[[ATP] + [ADP] + [AMP]]. Measurements were made on (1) fresh soil, extracted as soon as possible after field sampling (2) soil stored air-dry at 5°C for 18 days and (3) soil stored air-dry at 5°C for 57 days and then rewetted to the original field moisture content and incubated aerobically for 2.5 h at 10°C before extraction.In moist soil the biomass maintains both ATP and AEC at levels close to those of activity growing cells, even though little of the biomass in soil can be in active growth at any given time. ATP accounted for 77% of the total adenine nucleotides (A_T) in the fresh soil, with an AEC of 0.85 (a value comparable to that found in microorganisms undergoing active growth in vitro. In contrast, ATP only accounted for 28% of A_T in the air-dried soil, with an AEC of 0.46. When the air-dried soil was rewetted, ATP increased to 66% of A_T and the AEC increased to 0.76. However, A_T in the air-dried soil (7.65 nmol g^?1 soil) was of the same order as that in rewetted soil (6.70 nmol g^?1) even though the AEC's were very different.These results show that the soil microbial biomass does not maintain a high AEC when air-dried. Once remoistened, the population tends to restore its AEC to the original value. This restoration occurs so rapidly that it cannot be due to the formation of a new biomass.     

Specific measurement of soil microbial ATP

A direct procedure to extract and determine microbial adenosine 5'-triphosphate (ATP) in soil has been worked out. The soil is homogenized in cold Tris-EDTA-NaN₃ (TEA) buffer. The ATP content of 100 μl of a 1/1000 suspension is directly determined in a photoncountcr after addition of a detergent (NRB^®) extractant and the firefly luciferin-luciferase system. The method was tested on a wide variety of organisms and soils and compared to several other methods to determine ATP in soils. The method gives ATP recoveries of a minimum of 80% upon addition of cultures to soils.Furthermore, it is rapid, applicable to all soils examined, and most of all strictly specific for microbial biomass.     

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.     

A method for measuring adenosine triphosphate in soil

A method was devised for the extraction and measurement of adenosine 5'-triphosphate (ATP) in soil that minimizes sorption of ATP on the soil colloids. Soil was ultrasonified for 1 min with a solution containing trichloracetic acid (0.5 m). disodium hydrogen orthophosphate (0.25 m) and paraquat dichloride (0.1 m). The ATP content of the filtered extract was determined without further treatment in a scintillation spectrometer by the firefly luciferin-luciferase system. Recovery of added ATP was greater using the extratant containing trichloracetic acid, orthophosphate and paraquat than with trichloracetic acid alone or with a sulphuric acid extradant. Recoveries of added ATP ranged from 45% to 84% in thirteen different soils; ATP contents from 0.64 to 9.03 μg g^?1 soil.     

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.     

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.     

ATP content of soils estimated by two contrasting extraction methods

The ATP content of a series of soils was investigated in relation to various soil properties. Special attention was paid to the discrepancy in ATP, as estimated after extracting with TEA/NRB and TCA. It appears that the first procedure particularly relates to active microbial cells but extracts rather poorly certain types of older microbial biomass. Nevertheless, the TEA/NRB ATP values correlate very significantly with total soil microbial biomass as determined by the CHCl₃ fumigation method. In soils with active growing microbial biomass, the TEA/NRB and the TCA ATP values are about equal. In normal equilibrated soils the TEA/NRB ATP levels average about 40% of the total soil ATP levels. Finally, in densely rooted soils, the TCA ATP levels surpass largely the TEA/NRB levels, but they appear to a major extent to be due to plant cells.     

Relation between respiration,ATP content,and Adenylate Energy Charge (AEC) after incubation at different temperatures and after drying and rewetting

Temperature, drying, and rewetting are important climatic factors that control microbial properties. In the present study we looked at the respiration rates, adenosine 5′‐triphosphate (ATP) content, and adenylate energy charge (AEC) as a measure for energy status of microbial biomass in the upper 5 cm of mineral soils of three beech forests at different temperatures and after rewetting. The soils differed widely in pH (4.0 to 6.0), microbial biomass C (92 to 916 μg (g DW)^—1) and ATP content (2.17 to 7.29 nmol ATP (g DW)^—1). The soils were incubated for three weeks at 7 °C, 14 °C, and 21 °C. After three weeks the microbial properties were determined, retaining temperature conditions. The temperature treatment did not significantly affect AEC or ATP content, but respiration rates increased significantly with increasing temperature. In a second experiment the soils were dried for 12 hours at 40 °C. Afterwards the soils were rewetted and microbial properties were monitored for 72 hours. After the drying, respiration rates dropped below the detection limit, but within one hour after rewetting respiration rates increased above control level. Drying reduced AEC by 16 % to 44 % and ATP content by 47 % to 78 %, respectively. Rewetting increased AEC and ATP content significantly as compared to dry soil, but after 72 hours the level of the controls was still not reached. The level of AEC values indicated dormant cells, but ATP content increased. These results indicate that the microbial carbon turnover was not directly linked to microbial growth or microbial energy status. Furthermore our results indicate that AEC may describe an average energy status but does not reflect phases of growing, dormant, or dying cells in the complex microbial populations of soils.     

Interference from plant roots in the estimation of soil microbial ATP,C, N and P

Excised, solution-grown roots of maize or ryegrass added to two pasture soils at the rate of 6.0mg g^?1 and 13.8 mg g^?, respectively, increased the flush (fumigated minus control values) of CO₂-C by up to 1.89-fold, KCl extractable N by up to 1.88-fold, and NaHCO₃ extractable P by 3.28-fold. The ATP content of the soil was increased by up to 1.42-fold. Because of high variability the effect of the roots on the C and N flushes was not significant at P < 0.05.Incubation of the root-amended soils for 7 days at 25°C prior to fumigation much decreased the contribution from the roots to the C and N flush, and to the ATP content. There was, however, still a large significant effect of the roots on the P-flush, this being up to 3 times greater than the equivalent soil without roots.In soil samples with a high viable root density (> 6mg g^?1) such as may occur in dense pastures, greenhouse pot experiments or rhizosphere soil samples, it is recommended that they be incubated for 7 days prior to fumigation and analyses. Without such prior incubation there is the risk that root material may be included in the “microbial” biomass estimations.     

Immobilization and mineralization of nitrogen in soils incubated with pig slurry or ammonium sulphate

Nitrogen mineralization and immobilization were investigated in two soils incubated with ammonium sulphate or pig slurry over a range of temperatures and moisture contents. A reduction in the mineralization of soil organic N was observed in soils incubated with 100 μg NH₄⁺-Ng^?1 soil as ammonium sulphate at 30°C but not at lower temperatures. Addition of 100 μg NH₄⁺-N g^?1 soil as pig slurry resulted in a period of nett immobilization lasting up to 30 days at 5°C. Although the length of the immobilization phase was shorter at higher temperatures the total N immobilized was similar. The subsequent rate of mineralization in slurry-treated soils was not significantly greater (P = 0.05) than in untreated soils. There was no evidence of any subsequent increased mineralization arising from the immobilized N or slurry organic N for up to 175 days. The rate of immobilization was found to increase with increasing moisture content, though the period of nett immobilization was shorter, so that the amount of N immobilized was similar over a range of moisture contents from 10 to 40%. Approximately 40% of the NH₄⁺-N in the slurry was immobilized under the incubation conditions used.     

Phosphorus in the soil microbial biomass

Phosphorus in the soil microbial biomass (biomass P) and soil biomass carbon (biomass C) were linearly related in 15 soils (8 grassland, 6 arable, 1 deciduous woodland), with a mean P concentration of 3.3% in the soil biomass. The regression accounted for 82% of the variance in the data. The relationship was less close than that previously measured between soil biomass C and soil ATP content and indicates that biomass P measurements can only provide a rough estimate of biomass C content. Neither P concentration in the soil biomass, nor the amount of biomass P in soil, were correlated with soil NaHCO₃-extractable inorganic, organic or total P.The calculated mean annual flux of P through the biomass (in a soil depth of 10 cm) in 8 grassland soils was large, 23 kg P ha^?1 yr^?1, and more than three times the mean annual P flux through 6 arable soils (7 kg P ha^?1 yr^?1), suggesting that biomass P could make a significant contribution to plant P nutrition in grassland.About 3% of the total soil organic P in the arable soils was in microbial biomass and from 5 to 24% in the grassland soils. The decline in biomass P when an old grassland soil was put into an arable rotation for about 20 yr was sufficient to account for about 50% of the decline in total soil organic P during this period. When an old arable soil reverted to woodland, soil organic P doubled in 100 yr; biomass P increased 11-fold during the same period.     

An improved method for determination of adenosine triphosphate (ATP) in soil

Different methods for extraction of ATP from soil were examined. The methods were compared with respect to the efficiency of extraction of ATP from soil, and the content of ATP in the extracts was measured by the luciferin-luciferase system. The following extraction procedure was found to be the most efficient:Shaking of the soil with sulphuric acid, followed by filtration, cation exchange of the extract on Na⁺ resin, and adjustment to pH 9.8 with ethanolamine.The method is simpler, faster and more convenient for processing large numbers of samples, than the earlier-described acid extraction methods. Furthermore, ATP levels of 5ngATPg^?1 dry wt soil can be measured and results can be reproduced within ± 5%.The respiration rates of the soils were also measured, and a high degree of correlation was found between carbon dioxide production and ATP levels determined by extraction with sulphuric acid or dimethylsulfoxid.     

Influence of storage on soil microbial biomass estimated by three biochemical procedures

The effects of 28 and 56 days' storage at 25°, 4° and ?20°C on the microbial biomass content of four soils from tussock grasslands were studied by three biochemical procedures. Two of the procedures involved measurement of CO₂ and mineral-N (Min-N) production by chloroform-fumigated and unfumigated soil, and consequent estimation of biomass C and Min-N flush respectively. In the third, adenosine 5'-triphosphate (ATP) content was determined.Patterns of CO₂ production were often influenced by storage treatment. The use of fixed incubation periods for estimating the CO₂ flush of fumigated soil and the steady rate of CO₂ production by unfumigated soil did, however, give biomass C estimates that were generally similar to those calculated from individually determined incubation periods for each treatment and soil.Biomass C values could change significantly at all storage temperatures, but generally least at ?20°C. Storage at ?20°C was also the most suitable for retaining ATP contents, whereas 4°C was best for values of Min-N flush. Values of Min-N flush after storage of soil at ?20°C decreased significantly in two of the soils but increased in another. No storage temperature was thus satisfactory for all three indices of microbial biomass. Generally, however, 4°C was adequate for short periods, and 25°C the least suitable.     

Methods to evaluate pesticide damage to the biomass of the soil microflora

Respiratory methods to estimate the amount of C in the soil microbial biomass and the relative contributions of procaryotes and eucaryotes to the biomass were used to evaluate the influence of pesticides on the soil microflora. Experiments were conducted with 5 and 50 μg·g^?1 of three fungicides, captan, thiram and verdasan. At 5 μg·g^?1 they caused significant decreases (40%) in the biomass; the organomercury fungicide verdasan also caused a shift from fungal to bacterial dominance. Within 8 days, biomass in captan- and thiram-amended soils had recovered to that of the controls. Although the fungal to bacterial balance was restored in verdasan-amended soils, biomass recovery was not complete. At 50 μg·g^?1 the fungicides caused long-term decreases in the biomass and altered the relative proportions of the bacterial and fungal populations. Verdasan had the greatest effect on soil microbial biomass and composition.     

Persistence of effects of nitrification inhibitors added to soils

Abstract

The persistence of the effects of four nitrification inhibitors (2‐ethynylpyridine, nitrapyrin, etridiazole, 3‐methylpyrazole‐l‐carboxamide) on nitrification in soil was assessed by measuring the ability of two soils to nitrify NH₄ ⁺ [added as (NH₄)₂SO₄] after they had been treated with 5 μg inhibitor g^‐1 soil and incubated at 10, 20, or 30°C for 0, 21, 42, 84, 126, or 168 days. The soils used differed markedly in organic‐matter content (1.2 and 4.2% organic C). The data obtained showed that the persistence of the effects of the inhibitors studied decreased markedly with increase in soil temperature from 10 to 30°C and that, whereas the initial inhibitory effects of the test compounds on nitrification were greatest with the soil having the lower organic‐matter content, the persistence of their effects at 20 or 30°C was greatest with the soil having the higher organic‐matter content. The inhibitory effects of 2‐ethynylpyridine and etridiazole on nitrification were considerably more persistent than those of nitrapyrin or 3‐methylpyrazole‐l‐carboxamide and were significant even after incubation of inhibitor‐treated soil at 20°C for 168 days.     

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)].