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.     

Impact of cycloheximide addition on adenylates in soil

Cycloheximide inhibits specifically the ribosomal protein synthesis of eukaryotic cells, i.e. the metabolism of soil fungi. We measured cycloheximide effects on adenylates in 20 different soils (0-10 cm depth) from arable, grass and forest land with a large variety of soil properties. The aims were (1) to assess the interactions between cycloheximide effects and soil properties and (2) to prove the relationship between cycloheximide effects on ATP and the ergosterol-to-microbial biomass C ratio, which is an indicator for the fungal proportion of the total microbial biomass. The adenylates ATP, ADP and AMP were measured 6 h after adding either 10 mg cycloheximide per gram soil in combination with 24 mg talcum per gram soil or 24 mg talcum per gram soil solely. The medians of the relative increases in AMP and ADP were 45 and 25% and the medians of the relative decreases in ATP and adenylates were −36 and −12%. These changes in adenylate composition lead to a cycloheximide-induced relative decrease in the adenylate energy charge level of 15%. The relative decrease in ATP content after cycloheximide addition was significantly correlated with the ATP-to-microbial biomass C ratio, but not with the ergosterol-to-microbial biomass C ratio. The absolute increase in ADP and the absolute decrease in ATP were affected by the clay content according to principal component analysis. The reduction of the ATP-to-microbial biomass C ratio indicates that this ratio had the potential of being an important ecotoxicological indicator of direct toxic effects of organic pollutants on soil microorganisms.     

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.     

Respiration pattern and microbial use of field-grown transgenic Bt-maize residues

A 49-day incubation experiment was carried out with the addition of field-grown maize stem and leaf residues to soil at three different temperatures (5, 15, and 25 °C). The aim was to study the effects of two transgenic Bt-maize varieties in comparison to their two parental non-Bt varieties on the mineralization of the residues, on their incorporation into the microbial biomass and on changes in the microbial community structure. The stem and leaf residues of Novelis-Bt contained 3.9 μg g⁻¹ dry weight of the Bt toxin Cry1Ab and those of Valmont-Bt only 0.8 μg g⁻¹. The residues of the two parental non-Bt varieties Nobilis and Prelude contained higher concentrations of ergosterol (+220%) and glucosamine (+190%) and had a larger fungal C-to-bacterial C ratio (+240%) than the two Bt varieties. After adding the Bt residues, an initial peak in respiration of an extra 700 μg CO₂-C g⁻¹ soil or 4% of the added amount was observed in comparison to the two non-Bt varieties at all three temperatures. On average of the four varieties, 19-38% of the maize C added was mineralized during the 49-day incubation at the three different temperatures. The overall mean increase in total maize-derived CO₂ evolution corresponded to a Q₁₀ value of 1.4 for both temperature steps, i.e. from 5 to 15 °C and from 15 to 25 °C. The addition of maize residues led to a strong increase in all microbial properties analyzed. The highest contents were always measured at 5 °C and the lowest at 25 °C. The variety-specific contents of microbial biomass C, biomass N, ATP and adenylates increased in the order Novelis-Bt ? Prelude<Valmont-Bt ? Nobilis. The mineralization of Novelis-Bt residues with the highest Bt concentration and lowest N concentration and their incorporation into the microbial biomass was significantly reduced compared to the parental non-Bt variety Nobilis. These negative effects increased considerably from 5 to 25 °C. The transgenic Bt variety Valmont did not show further significant effects except for the initial peak in respiration at any temperature.     

Microbial reaction of secondary tropical forest soils to the addition of leaf litter

Two soils from a secondary tropical forest at La Union, Philippines, predominantly vegetated with Swietenia marcrophylla and Gmelina arborea were amended with different leaf litter types (Eucalyptus camaldulensis, S. macrophylla, G. arborea, and Calliandra calothyrsus) and incubated in the laboratory for 49 days at 25 °C. The experiment was carried out to elucidate the reasons for a low ATP-to-microbial biomass C ratio and a high microbial biomass C-to-N ratio. This has been measured repeatedly in tropical forest soils. In the non-amended soils, the microbial biomass C-to-N ratio of 12.1 exceeded the soil organic C-to-total N ratio of 11, while the ergosterol-to-microbial biomass C ratio of 0.14% and the ATP-to-microbial biomass C ratio of 4.1 μmol g⁻¹ were both low. At the end of the incubation, the addition of the different leaf litter types led generally to a decrease in the microbial biomass C-to-N ratio and to an increase in the ATP-to-microbial biomass C ratio, adenylate energy charge (AEC) and especially to an increase in the ergosterol-to-microbial biomass C ratio. The increase in the ATP-to-microbial biomass C ratio and the decrease in the microbial biomass C-to-N ratio were positively related to the N concentration in the leaf litter, the increase in the ergosterol-to-microbial biomass ratio negatively. The reasons for a low ATP-to-microbial biomass C ratio and a high microbial biomass C-to-N ratio are P deficiency and probably a reduced access of soil microorganisms to N containing organic components at low soil organic C levels.     

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.     

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.     

Soil microbial properties down the profile of a black earth burie by colluvium

The activity and biomass of soil microorganisms were determined in samples at 0—140 cm depth taken from an arable site, where the soil has been developed by erosion and colluvial deposition overlaying a black earth at 70—110 cm depth. The central aim was to get an insight into the breakdown of increasingly old and thus recalcitrant soil organic matter down the profile, effects on the availability of C to microorganisms and the microbial community structure. From 0 to 140 cm depth, microbial biomass C decreased by 96%, biomass N by 97%, the adenylates ATP, ADP, and AMP as well as the basal respiration rate by 89%. No ergosterol was measured at 120—140 cm depth. All soil biological properties decreased in distinct steps after 30 cm and 50 cm depth. At 30—90 cm depth, the amounts of soil organic C and microbial biomass C per hectare of the present colluvium exceeded nearly three‐fold those in undisturbed aeolian loess sediments. The cation exchange significantly affected the relationships between microbial biomass C, biomass N, and the adenylates. As a consequence, none of the ratios between the soil microbial biomass properties revealed constant gradients throughout the profile. The adenylate energy charge (AEC) varied between the different soil layers insignificantly around a mean of 0.71. It was the most stable ratio down the profile showing absolutely no depth gradient, the lowest depth‐to‐depth variation, and also the lowest within depth variability. The other ratios between soil organic C, basal respiration, ergosterol, microbial biomass C and biomass N also did not reveal any marked changes in the microbial community structure.     

Estimation of conversion factors for fungal biomass determination in compost using ergosterol and PLFA 18:2ω6,9

Eleven species of common fungi from compost were analysed for their content of ergosterol and phospholipid fatty acids (PLFAs) after growth on agar media. Mean content of ergosterol was 3.1 mg g⁻¹ dw of fungal mycelium (range 1-24 mg g⁻¹ dw). Total amount of PLFAs varied between 2.6 and 43.5 μmol g⁻¹ dw of fungi (mean 14.9 μmol g⁻¹ dw). The most common PLFAs were 16:0, 18:2ω6,9 and 18:1ω9, comprising between 79 and 97 mol% of the total amount of PLFAs. The PLFA 18:2ω6,9, suggested as a marker molecule for fungi, comprised between 36 and 61 mol% of the total PLFAs in the Ascomycetes, between 45 and 57 mol% in the Basidiomycetes and 12-22 mol% in the Zygomycetes. There was a good correlation between the content of the two fungal marker molecules, ergosterol and the PLFA 18:2ω6,9, with a mean content of 1 mg ergosterol being equivalent to 2.1 μmol of 18:2ω6,9. Based on results from the fungal isolates, conversion factors were calculated (5.4 mg ergosterol g⁻¹ biomass C and 11.8 μmol 18:2ω6,9 g⁻¹ biomass C) and applied to compost samples in which both the ergosterol and the PLFA 18:2ω6,9 content had been measured. This resulted in similar estimates of fungal biomass C using the two marker molecules, but was three to five times higher than total microbial biomass C calculated using ATP content in the compost. This could partly be explained by the fact that both of the markers used for fungal biomass are cell membrane constituents. Thus, the ergosterol and the PLFA content were related to the hyphal diameter of the fungi, where fungi with thinner hyphae had higher ergosterol concentrations than fungi with thicker hyphae. This could also partly explain the large interspecific variation in content of the two marker molecules.     

Dynamics of microbial biomass and nitrogen supply during primary succession on blastfurnace slag dumps in dry tropics

This paper reports the role of microbial biomass in the establishment of N pools in the substratum during primary succession (till 40-year age) in Blastfurnace Slag Dumps, an anthropogenically created land form in the tropics. Initially in the depressions in the slag dumps fine soil particles (silt+clay) accumulate, retaining moisture therein, and providing microsites for the accumulation of microbial biomass. In all sites microbial biomass showed distinct seasonality, with summer-peak and rainy season-low standing crops. During the summer season microbial biomass C ranged from 18.6 μg g⁻¹ in the 1-year old site to ca. 235 μg g⁻¹ in the 40-year old site; correspondingly, microbial biomass N ranged from 1.22 to 40 μg g⁻¹. On sites 2.5-years of age and younger, the microbial biomass N content accounted for more than 50% of the organic N in the soil, whereas the proportion of microbial biomass N was ca. 7% of organic N in 40-year old site. The strong correlation between microbial biomass and total N in soil indicated a significant role of microbes in the build-up of nitrogen during the initial stages of succession in the slag dumps. Though the organic N pool in the soil was low (594 mg kg⁻¹) even after 40 years of succession, the available N (NH₄-N and NO₃-N) contents in the soil were generally high through the entire age series (ca. 16-32 μg g⁻¹) during the rainy season (which supports active growth of the herbaceous community). The high mineral-N status on the slag dump was related with high N-mineralization rates, particularly in the young sites (20.6 and 13.9 μg g⁻¹ month⁻¹ at 1 and 2.5-year age). We suggest that along with the abiotic factors having strong effect on ecosystem functioning, the microbial biomass, an important biotic factor, shows considerable influence on soil nutrient build-up during early stages of primary succession on the slag dumps. The microbial biomass dynamics initiates biotic control in developing slag dumps ecosystem through its effect on nitrogen pools and availability.     

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.     

Soil microbial and nutrient dynamics in a wet Arctic sedge meadow in late winter and early spring

Microbial activity is known to continue during the winter months in cold alpine and Arctic soils often resulting in high microbial biomass. Complex soil nutrient dynamics characterize the transition when soil temperatures approach and exceed 0 °C in spring. At the time of this transition in alphine soils microbial biomass declines dramatically together with soil pools of available nutrients. This pattern of change characterizes alpine soils at the winter-spring transition but whether a similar pattern occurs in Arctic soils, which are colder, is unclear. In this study amounts of microbial biomass and the availability of carbon (C), nitrogen (N) and phosphorus (P) for microbial and plant growth in wet peaty soils of an Arctic sedge meadow have been determined across the winter-spring boundary. The objective was to determine the likely causes of the decline in microbial biomass in relation to temperature change and nutrient availability. The pattern of soil temperature at depths of 5-15 cm can be divided into three phases: below −10 °C in late winter, from −7 to 0 °C for 7 weeks during a period of freeze-thaw cycles and above 0 °C in early spring. Peak microbial biomass and nutrient availability occurred early in the freeze-thaw phase. Subsequently, a steady decrease in inorganic N occurred, so that when soil temperatures rose above 0 °C, pools of inorganic nutrients in soils were very low. In contrast, amounts of microbial C and soluble organic C and N remained high until the end of the period of freeze-thaw cycles, when a sudden collapse occurred in soluble organic C and N and in phosphatase activity, followed by a crash in microbial biomass just prior to soil temperatures rising consistently above 0 °C. Following this, there was no large pulse of available nutrients, implying that competition for nutrients from roots results in the collapse of the microbial pool.     

Microbial community response to nitrogen deposition in northern forest ecosystems

The productivity of temperate forests is often limited by soil N availability, suggesting that elevated atmospheric N deposition could increase ecosystem C storage. However, the magnitude of this increase is dependent on rates of soil organic matter formation as well as rates of plant production. Nonetheless, we have a limited understanding of the potential for atmospheric N deposition to alter microbial activity in soil, and hence rates of soil organic matter formation. Because high levels of inorganic N suppress lignin oxidation by white rot basidiomycetes and generally enhance cellulose hydrolysis, we hypothesized that atmospheric N deposition would alter microbial decomposition in a manner that was consistent with changes in enzyme activity and shift decomposition from fungi to less efficient bacteria. To test our idea, we experimentally manipulated atmospheric N deposition (0, 30 and 80 kg NO₃⁻-N) in three northern temperate forests (black oak/white oak (BOWO), sugar maple/red oak (SMRO), and sugar maple/basswood (SMBW)). After one year, we measured the activity of ligninolytic and cellulolytic soil enzymes, and traced the fate of lignin and cellulose breakdown products (¹³C-vanillin, catechol and cellobiose).In the BOWO ecosystem, the highest level of N deposition tended to reduce phenol oxidase activity (131±13 versus 104±5 μmol h⁻¹ g⁻¹) and peroxidase activity (210±26 versus 190±21 μmol h⁻¹ g⁻¹) and it reduced ¹³C-vanillin and ¹³C-catechol degradation and the incorporation of ¹³C into fungal phospholipids (p<0.05). Conversely, in the SMRO and SMBW ecosystems, N deposition tended to increase phenol oxidase and peroxidase activities and increased vanillin and catechol degradation and the incorporation of isotope into fungal phospholipids (p<0.05). We observed no effect of experimental N deposition on the degradation of ¹³C-cellulose, although cellulase activity showed a small and marginally significant increase (p<0.10). The ecosystem-specific response of microbial activity and soil C cycling to experimental N addition indicates that accurate prediction of soil C storage requires a better understanding of the physiological response of microbial communities to atmospheric N deposition.     

Elevated enzyme activities in soils under the invasive nitrogen-fixing tree Falcataria moluccana

Like other N-fixing invasive species in Hawaii, Falcataria moluccana dramatically alters forest structure, litterfall quality and quantity, and nutrient dynamics. We hypothesized that these biogeochemical changes would also affect the soil microbial community and the extracellular enzymes responsible for carbon and nutrient mineralization. Across three sites differing in substrate texture and age (50-300 years old), we measured soil enzyme activities and microbial community parameters in native-dominated and Falcataria-invaded plots. Falcataria invasion increased acid phosphatase (AP) activities to >90 μmol g⁻¹ soil h⁻¹ compared to 30-60 μmol g⁻¹ soil h⁻¹ in native-dominated stands. Extracellular enzymes that mineralize carbon and nitrogen also increased significantly under Falcataria on the younger substrates. By contrast, total microbial biomass and mycorrhizal abundance changed little with invasion or substrate. However, fungal:bacterial ratios declined dramatically with invasion, from 2.69 and 1.35 to <0.89 on the 50- and 200-year-old substrates, respectively. These results suggest that Falcataria invasion alters the composition and function of belowground soil communities in addition to forest structure and biogeochemistry. The increased activities of AP and other enzymes that we observed are consistent with a shift toward phosphorus limitation and rapid microbial processing of litterfall C and N following Falcataria invasion.     

Soil biochemical and microbial indices in wet tropical forests: Effects of deforestation and cultivation

Little information is available about the long‐term effects of deforestation and cultivation on biochemical and microbial properties in wet tropical forest soils. In this study, we evaluated the general and specific biochemical properties of soils under evergreen, semi‐evergreen, and moist deciduous forests and adjacent plantations of coconut, arecanut, and rubber, established by clear felling portions of these forests. We also examined the effects of change in land use on microbial indices and their interrelationships in soils. Significant differences between the sites occurred for the biochemical properties reflecting soil microbial activity. Microbial biomass C, biomass N, soil respiration, N mineralization capacity, ergosterol, levels of adenylates (ATP, AMP, ADP), and activities of dehydrogenase and catalase were, in general, significantly higher under the forests than under the plantations. Likewise, the activities of various hydrolytic enzymes such as acid phosphomonoesterase, phosphodiesterase, casein‐protease, BAA‐protease, β‐glucosidase, CM‐cellulase, invertase, urease, and arylsulfatase were significantly higher in the forest soils which suggested that deforestation and cultivation markedly reduced microbial activity, enzyme synthesis and accumulation due to decreased C turnover and nutrient availability. While the ratios of microbial biomass C : N and microbial biomass C : organic C did not vary significantly between the sites, the ratios of ergosterol : biomass C and ATP : biomass C, qCO2 and AEC (Adenylate Energy Charge) levels were significantly higher in the forest sites indicating high energy requirements of soil microbes at these sites.     

Long-term effects of organic farming on fungal and bacterial residues in relation to microbial energy metabolism

Samples from the bio-dynamic, bio-organic, and conventional trial, Therwil, Switzerland, were analyzed with the aim of determining the effects of organic land use management on the energy metabolism of the soil microbial biomass and on the fraction of microbial residues. The contents of adenylates, adenosine triphosphate (ATP), glucosamine, muramic acid, and galactosamine were significantly largest in the biodynamic organic farming (BYODIN) treatment and significantly lowest in the conventional farming treatment with inorganic fertilization (CONMIN). In contrast, the ergosterol-to-ATP ratio and fungal C-to-bacterial C ratios were significantly lowest in the BYODIN treatment and significantly largest in the CONMIN treatment. No clear treatment effects were observed for the ergosterol content and the adenylate energy charge (AEC), the ATP-to-microbial biomass C ratio and the ergosterol-to-fungal C ratio. Ergosterol, an indicator for saprotrophic fungal biomass, and fungal residues were significantly correlated. The microbial biomass carbon-to-nitrogen ratio showed a negative relationship with the AEC and strong positive relationships with the ratios ergosterol-to-microbial biomass C, ergosterol-to-ATP and fungal C-to-bacterial C. In conclusion, the long-term application of farmyard manure in combination with organic farming practices led to an increased accumulation of bacterial residues.     

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 colonisation of roots as a function of plant species

Fifteen plants species were grown in the greenhouse on the same soil and sampled at flowering to obtain rhizosphere soil and root material. In both fractions, the data on fungal and bacterial tissue obtained by amino sugar analysis were compared with the total microbial biomass based on fumigation-extraction and ergosterol data. The available literature on glucosamine concentrations in fungi and on muramic acid concentrations in bacteria was reviewed to prove the possibility of generating conversion values for general use in root material. All microbial properties analysed revealed strong species-specific differences in microbial colonisation of plant roots. The root material contained considerable amounts of microbial biomass C and biomass N, reaching mean levels of 10.9 and 1.4 mg g⁻¹ dry weight, respectively. However, the majority of CHCl₃ labile C and N, i.e. 89 and 55% was root derived. The average amount of ergosterol was 13 μg g⁻¹ dry weight and varied between 0.0 for Phacelia roots and 45.5 μg g⁻¹ dry weight for Vicia roots. The ergosterol content in root material of mycorrhizal and non-mycorrhizal plant species did not differ significantly. Fungal glucosamine was converted to fungal C by multiplication by 9 giving a range of 7.1-25.9 mg g⁻¹ dry weight in the root material. Fungal C and ergosterol were significantly correlated. Bacterial C was calculated by multiplying muramic acid by 45 giving a range from 1.7 to 21.6 mg g⁻¹ dry weight in the root material. In the root material of the 15 plant species, the ratio of fungal C-to-bacterial C ranged from 1.0 in mycorrhizal Trifolium roots to 9.5 in non-mycorrhizal Lupinus roots and it was on average 3.1. These figures mean that the microbial tissue in the root material consists on average of 76% fungal C and 24% bacterial C. The differences in microbial colonisation of the roots were reflected by differences in microbial indices found in the rhizosphere soil, most strongly for microbial biomass C and ergosterol, but to some extent also for glucosamine and muramic acid.     

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.     

Measuring microbial uptake of nitrogen in nutrient-amended sandy soils—A mass-balance based approach

Soil microbial biomass N is commonly determined through fumigation-extraction (FE), and a conversion factor (K_EN) is necessary to convert extractable N to actual soil biomass N. Estimation of K_EN has been constrained by various uncertainties including potential microbial immobilisation. We developed a mass-balance approach to quantify changes in microbial N storage during nutrient-amended incubation, in which microbial uptake is determined as the residual in a ‘mass-balance’ based on soil-water N before and after amended incubation. The approach was applied to three sandy soils of southwestern Australia, to determine microbial N immobilisation during 5-day incubation in response to supply of 2.323 mg C g⁻¹, 100 μg N g⁻¹ and 20 μg P g⁻¹. The net N immobilisation was estimated to be 95-114 μg N g⁻¹ in the three soils, equivalent to 82.7-85.1% of soil-water N following the amendment. Such estimation for microbial uptake does not depend on fumigation and K_EN conversion, but for comparison purposes we estimated ‘nominal’ K_EN values (0.11-0.14) for the three soils, which were comparable to previously reported K_EN from soils receiving C and N amendment. The accuracy of our approach depends on the mass-balance equation and the integrated measurement errors of the multiple N pools, and was assessed practically through recoveries of added-N when microbial uptake can be minimised. Near-satisfactory recoveries were achieved under such conditions. Our mass-balance approach provides information not only about changes in the microbial biomass nitrogen storage, but also major N-pools and their fluxes in regulating soil N concentrations under substrate and nutrient amended conditions.