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.     

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.     

Adenylate energy charge and ATP-to-microbial biomass C ratio in soils differing in the intensity of disturbance

We carried out an 8-days' incubation experiment with three different intensities of soil disturbance to analyse the effects on the ATP-to-microbial biomass C ratio and on the adenylate energy charge (AEC=(ATP+0.5×ADP)/(AMP+ADP+ATP). Single mixing of soil at 50% water holding capacity with a spatula during weighing of the samples into extraction jars at the end of the 8-days' incubation or 8-times repeated daily mixing for 2 min triggered the immediate formation of ATP, increasing both AEC and the ATP-to-microbial biomass C ratio. The energy for this extra ATP produced seems to be mainly derived from an accelerated turnover of C within the microbial biomass. In contrast, 8-days' continuous mixing led to a significant decrease in AEC and ATP-to-microbial biomass C ratio.     

Specific respiration rates, adenylates, and energy budgets of soil microorganisms after addition of transgenic Bt-maize straw

An arable soil was incubated with straw (stem+leaves) of two transgenic Bt-maize varieties (Novelis: event MON810 and Valmont: event Bt176) and the two corresponding near-isogenic varieties (Nobilis and Prelude). The aim was to evaluate the use of these substrates for microbial growth and maintenance in soil during early decomposition. The addition of Bt-maize straw increased CO₂ production rates and the specific respiration rates CO₂-C/microbial biomass C and CO₂-C/ATP significantly compared with the addition of non-Bt maize straw. This extra energy in the Bt-maize straw could not be used for microbial biomass or ATP and ADP production, and was lost for maintenance. In addition, increased death rates of microbial biomass occurred in the soils treated with the Bt-maize straw from day 3 to 21. Generally, most of the energy was stored in microbial biomass, whereas only 10% of energy was stored in ATP, and only 1-2% in ADP. The AEC (adenylate energy charge: (ATP+0.5×ADP)/(AMP+ADP+ATP)) was not affected by any treatment. The reasons for the lower efficiency of microbial substrate use after adding Bt-maize straw cannot be fully explained by the present experiment. However, a risk assessment has to look at the impact of transgenic plant material on soil microorganisms at different maturity stages.     

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 as an estimate of microbial biomass C in different soil groups

Adenylate (i.e. adenosine tri- (ATP), di- (ADP) and monophosphates (AMP)) and microbial biomass C data were collected over a wide range of sites including forest floor layers and forest, grassland and arable soils. Microbial biomass C was measured by fumigation extraction and adenylates after alkaline Na₃PO₄/DMSO/EDTA extraction and HPLC detection. Our aims were (1) to test whether the sum of adenylates is a better estimate for microbial biomass than the determination of ATP, (2) to compare our conversion values with those proposed by others, and (3) to analyse whether soil properties or land use form affect the relationships between ATP, adenylates and microbial biomass C. A close relationship was found between microbial biomass C and ATP (r=0.96), but also with the sum of adenylates (r=0.96) within all appropriately conditioned soil samples (n=112). In the mineral soil (n=98), the geometric means of the ATP-to-microbial biomass C ratio and the adenylates-to-microbial biomass C ratio were 7.4 and 11.4 μmol g⁻¹, respectively. The mean ratios did not differ significantly between the different texture classes and land use forms. In the forest floor, the ATP-to-microbial biomass C ratio and the adenylates-to-microbial biomass C ratio were both roughly two-thirds of those of the mineral soil. The average adenylate energy charge (AEC) of all soil samples was 0.79 and showed a strong negative relationship with the soil pH (r=−0.69). However, the AEC is presumably only indirectly affected by the soil pH.     

Reaction of microorganisms to rewetting in continuous cereal and legume rotation soils of semi-arid Sub-Saharan Africa

A 20-day incubation experiment with continuous cereal (CC) versus cereal legume (CL) rotation soils of two semi-arid Sub-Saharan sites (Fada-Kouaré in Burkina Faso, F, and Koukombo in Togo, K) were carried out to investigate the effects of rewetting on soil microbial properties. Site- and system-specific reactions of soil microorganisms were observed on cumulative CO₂ production, adenylates (ATP, ADP, and AMP), microbial biomass C and N, ergosterol, muramic acid and glucosamine. Higher values of all parameters were found in the CL rotation soils and in both soils from Fada-Kouaré. While the inorganic N concentration showed only a system-specific response to rewetting, the adenylate energy charge (AEC) showed only a site-specific response. ATP recovered within 6 h after rewetting from ADP and AMP due to rehydration of microorganisms and not due to microbial growth. Consequently, no N seemed to be immobilized by microorganisms and all NO₃ in the soil was immediately available to the plants. The fungal cell-membrane component ergosterol was three (CC) and five (CL) times larger at Fada than in the respective soils at Koukombo. The concentrations of the bacterial cell-wall component muramic acid were by 20% and of mainly fungal glucosamine by 10% larger in the CL rotation soils than in the CC soils. This indicates long-shifts in the microbial community structure.     

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.     

Long‐term effects of leguminous cover crops on microbial indices and their relationships in soils of a coconut plantation of a humid tropical region

In this study, leguminous crops like Atylosia scarabaeoides, Centrosema pubescens, Calopogonium mucunoides, and Pueraria phaseoloides. grown as soil cover individually in the interspaces of a 19‐yr‐old coconut plantation in S. Andaman (India) were assessed for their influence on various microbial indices (microbial biomass C, biomass N, basal respiration, ergosterol, levels of ATP, AMP, ADP) in soils (0–50 cm) collected from these plots after 10 years. The effects of these cover crops on . CO₂ (metabolic quotient), adenylate energy charge (AEC), and the ratios of various soil microbial properties viz., biomass C : soil organic C, biomass C : N, biomass N : total N, ergosterol : biomass C, and ATP : biomass C were also examined. Cover cropping markedly enhanced the levels of organic matter and microbial activity in soils after the 10‐yr‐period. Microbial biomass C and N, basal respiration, . CO₂, ergosterol and levels of ATP, AMP, ADP in the cover‐cropped plots significantly exceeded the corresponding values in the control plot. While the biomass C : N ratio tended to decrease, the ratios of biomass N : total N, ergosterol : biomass C, and ATP : biomass C increased significantly due to cover cropping. Greater ergosterol : biomass C ratio in the cover‐cropped plots indicated a decomposition pathway dominated by fungi, and high . CO₂ levels in these plots indicated a decrease in substrate use efficiency probably due to the dominance of fungi. The AEC levels ranged from 0.80 to 0.83 in the cover‐cropped plots, thereby reflecting greater microbial proliferation and activity. The ratios of various microbial and chemical properties could be assigned to three different factors by principal components analysis. The first factor (PC1) with strong loadings of ATP : biomass C ratio, AEC, and . CO₂ reflected the specific metabolic activity of soil microbes. The ratios of ergosterol : biomass C, soil organic C : total N, and biomass N : total N formed the second factor (PC2) indicating a decomposition pathway dominated by fungi. The biomass C : N and biomass C : soil organic C ratios formed the third principal component (PC3), reflecting soil organic matter availability in relation to nutrient availability. Overall, the study suggested that Pueraria phaseoloides. or Atylosia scarabaeoides were better suited as cover crops for the humid tropics due to their positive contribution to soil organic C, N, and microbial activity.     

Microbial biomass and activity indices after organic substrate addition to a selenium-contaminated soil

High concentrations of Se in soil might have negative effects on microorganisms. For this reason, the effect of organic substrate addition (glucose + maize straw) on Se volatilisation in relation to changes in microbial biomass and activity indices was investigated using an artificially Se-contaminated soil. Microbial biomass N was reduced on average by more than 50% after substrate addition, but adenylate energy charge (AEC) and metabolic quotient qCO₂ were both increased. The Se content decreased by nearly 30% only with the addition of the organic substrate at 25°C. No significant Se loss occurred without substrate at 25°C or with substrate at 5°C. In the two treatments with substrate addition, the substrate-derived CO₂ evolution was about 30% lower with Se addition than without. In contrast, Se had no effect on any of the other soil microbial indices analysed, i.e. microbial biomass C, microbial biomass N, adenosine triphosphate (ATP), AEC, ATP-to-microbial biomass C, and qCO₂.     

Phosphatase activity does not limit the microbial use of low molecular weight organic-P substrates in soil

Plant roots and soil microorganisms contain significant quantities of low molecular weight (MW) phosphorylated nucleosides and sugars. Consequently, upon death these can represent a significant input of organic-P to the soil. Some of these organic-P substrates must first be dephosphorylated by phosphatases before being assimilated by the soil microbial community while others can be taken up directly from soil solution. To determine whether sorption or phosphatase activity was limiting the bioavailability of low MW organic-P in soil we compared the microbial uptake and C mineralization of a range of ¹⁴C-labeled organic-P substrates [glucose-6-phosphate, adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP)] to that of the parent compounds (adenosine and glucose). In a fertile grassland soil we showed that at low organic-P substrate concentrations (<0.5 mM) phosphatase activity did not limit microbial uptake or mineralization in comparison to their non-phosphorylated counterparts. However, at high substrate concentrations (1-10 mM) the mineralization of the organic-P compounds was significantly lower than that of the non-phosphorylated compounds suggesting that phosphatase activity or microbial transporter capacity limited bioavailability. Sorption to the solid phase followed the series glucose<adenosine<G-6-P<AMP<ADP=ATP. However, sorption of the organic-P compounds to the solid phase did not appear to greatly affect bioavailability. The high adenosine mineralization capacity of the microbial biomass suggests that nucleosides may represent a significant source of C and N to the soil microbial biomass. We conclude that at low organic-P substrate concentrations typical of those in soil, neither phosphatase activity nor sorption greatly limits their bioavailability.     

Phosphorus fractions in poultry litter as affected by flue‐gas desulphurization gypsum and litter stacking

Previous research has shown that the addition of flue‐gas desulphurization (FGD) gypsum to poultry litter decreases water‐soluble P. No information is currently available, however, on extractable P fractions in poultry litter and P availability as affected by gypsum. The first objective of this work was to evaluate the effect of incubation time and rate of gypsum addition to litter alone or litter mixed with soil on total P and inorganic P in sequential extracts of H₂O, 0.5 m NaHCO₃, 0.1 m NaOH and 1 m HCl. Poultry litter was mixed with 25, 50, or 75% gypsum (by weight) and incubated alone or mixed with soil for 63–93 days at 25 °C, with periodic sequential extractions. For litter alone or litter mixed with soil, adding gypsum decreased total P and inorganic P in the H₂O fraction and increased both P forms in the NaHCO₃ fraction. These changes did not affect plant P availability as measured by Mehlich‐1 P. Increasing incubation time decreased total P and inorganic P in the H₂O fraction of litter alone or litter mixed with soil, which was apparently caused by P immobilization by fungi. A second objective of this study was to evaluate P in the H₂O and NaHCO₃ fractions of litter as affected by stacking time and depth. Litter was stacked to a height of 1.2 m with samples taken immediately after stacking and 31 days later to be sequentially extracted for total P and inorganic P. Stacking time did not affect P in the H₂O fraction, but it increased P in the NaHCO₃ fraction by 25%. These results suggest that stacking poultry litter may increase the amount of labile P.     

The effect of plant material on the relationship between sodium acetate and sodium bicarbonate extractable phosphorus in a Latahco silt loam soil

Abstract

Standardization of the P soil test procedures is desirable; however, both NaOAc and NaHCO₃ are currently used to extract P from soils in the Pacific Northwest region of the USA. The purpose of this study was to determine the relationship between NaOAc and NaHCO₃ extractable P in soils and to evaluate the effect of plant material on this relationship in a northern Idaho soil. The Ap horizon of a Latahco silt loam was used and alfalfa (Medicago sativa), pea (Pisum sativum) and wheat (Triticum aestivum) plant materials were added as amendments at rates of 0%, 1%, 5% and 10% (w/w). The soils were incubated for 20 weeks under controlled conditions. In addition, other parameters studied included soil water potential (‐0.05, ‐0.15 and ‐0.40 MPa), incubation temperature (10, 20 and 30°C and incubation period. P in samples was extracted by NaOAc and NaHCO₃ extractants. A statistically significant linear relationship between NaOAc and NaHCO₃ extractable P was observed (r² = 0.96). In addition, the types of plant residues added to soil differently affected P extraction by the two extractants. The difference between NaOAc and NaHCO₃ extractable P was greatest in the wheat material treatment while alfalfa material resulted in the smallest effect. Sodium acetate extractable P values increased faster than NaHCO₃ extractable P with increasing amendment rate.

A simple regression relationship will allow conversion between NaOAc and NaHCO₃ extractable P in the Latahco soil. Additions of less than 5 mt/ha plant material will have a minimal impact on this relationship.     

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.     

免耕和秸秆覆盖对黑垆土磷素形态组分的影响

[目的]探究免耕及添加秸秆条件下黑垆土土壤磷组分特征及其与AM真菌侵染的关系,了解雨养农业区农业系统磷素利用效率。[方法]在陇东黄土高原黑垆土区域,测定传统耕作、传统耕作+秸秆覆盖、免耕和免耕+秸秆覆盖4种处理小麦—玉米—大豆轮作系统中玉米阶段土壤全磷、速效磷组分及AM真菌菌根侵染率。[结果]水土保持耕作处理实施9a后,免耕和秸秆覆盖处理下0—5cm土壤磷素含量显著提高,活性磷组分H2O—Pi,NaHCO3—Pi,NaOH—Pi分别比对照提高84.6%,85.2%和56.6%;活性无机磷(H2O—Pi,NaHCO3—Pi之和)和潜在活性磷(NaOH—Pi)分别占总无机磷的11.4%和4.5%,全磷含量与磷组分、速效磷与磷组分呈显著正相关,2个免耕处理菌根侵染率分别比对照增加20.8%和16.5%。[结论]免耕和秸秆覆盖显著提高了土壤磷含量,免耕对AM真菌菌根侵染率有积极影响。     

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.     

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.     

Determination of phosphate in solution at different ionic composition using malachite green

Abstract

The malachite green method was sometimes used to determine low concentrations of inorganic phosphate due to its high sensitivity. The aim of this work was to test the suitability of this method for the determination of phosphorus (P) extracted by various reagents, e.g., KCl 0.01–1.20M, CaCl₂ 0.01–0.1M, Na₂SO₄ 0.01–0.40M, NaHCO₃ 0.1M at pH 8.5, and NaOH 0.1M+NaCl 1M. The malachite green method was also compared with the traditional molybdenum blue method on 35 soil extracts. Color development reached stability within 2 hrs and was stable for up to 24 hrs for dilute solutions. For concentrated solutions the stability was inversely proportional to the concentration of the reagent. Salt concentration appeared to have no effect on absorbance in KCl extracts of up to 1.2M and in Na₂SO₄ extracts of up to 0.05M. Higher concentrations of sodium sulfate induced flocculation and precipitation of the dye complex, as did CaCl₂ above 0.04 M. A strong correlation was found between the malachite green and the molybdenum blue method. The malachite green method can be used for P determination in soil extracts when appropriate time of color development is provided and salt concentration is taken into account.     

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.     

Influence of pH,dispersion methods,and different compounds on adenosine triphosphate recovery from soil

We studied the recovery of ATP from soil. A soil-water suspension was prepared by two different methods (simple stirring or ballottini mill treatment) at different pH levels and in the presence of different chemicals [Na₂SO₄, Na₃PO₄, Na₅P₃O₁₀, adenosine, ethylenediaminetetra-acetic acid (EDTA), TRIS]. The ATP recovery was evaluated by adding [³H]-8 ATP to the solution and comparing the values obtained by radioactivity measurements with those obtained by an enzymatic assay. Strongly acidic (pH lower than 1.0) or alkaline (pH 10.0) extractions yielded the best ATP recoveries compared with intermediate pH values. At pH 10.0, the addition of Na₃PO₄ or Na₅P₃O₁₀ gave a high level of ATP recovery, 68 and 96%, respectively. No ATP hydrolysis occurred under alkaline extraction conditions. Under acidic extraction conditions, the addition of adenosine, EDTA, Na₂SO₄, or Na₅P₃O₁₀ improved ATP recovery but it was never higher than 34%. The results were discussed in terms of the effects of different physical and chemical conditions on cell disruption, ATP stability, ATP interactions with soil components, and ATP solubilization.