Microbial immobilisation and turnover of N labelled substrates in two arable soils under field and laboratory conditions

Microbial biomass N dynamics were studied under field and laboratory conditions in soils of high yield (HY) and low yield (LY) areas in an agricultural field. The objective of the study was to determine the size and activity of soil microbial biomass in the soils of the different yield areas and to compare these data obtained under field and laboratory conditions. Soils were amended with ¹⁵N labelled mustard (Sinapis alba) residues (both experiments) and labelled nitrate (laboratory only) at 30 μg N g⁻¹ dry soil. Soil microbial biomass (SMB) N, mineral N (N_min) and total N content was monitored both in the field and in the laboratory. N₂O efflux was additionally measured in laboratory treatments. Isotope ratios were determined for SMB in both experiments, for all other parameters only in the laboratory treatments. In the laboratory less amounts of added substrate N were immobilised by the SMB in HY soils compared to LY soils, whereas in the field immobilisation of added N by SMB was higher in HY soils initially and slightly lower after 40 days of incubation. Calculated turnover times in the laboratory nitrate, laboratory mustard and field mustard amendments were 0.18, 0.27 and 0.74 years (HY) and 0.22, 0.61 and 1.01 years (LY), respectively. The turnover times of added substrate N always showed the trend to be faster in HY soils compared to LY soils. A faster turnover of nutrients in the HY soils may involve a better nutrient supply of the plants, which coincides with the higher agricultural yield observed in these areas.     

Decomposition and distribution of N labelled mustard litter (Sinapis alba) in physical soil fractions of a cropland with high- and low-yield field areas

High-yield (HY) areas of an agricultural cropland were characterized by different positions on a slope and lower silt and clay contents, compared to low-yield (LY) areas, and this was associated with differences in water regime and C and N turnover. To understand differences in N flows of HY and LY areas, a combination of ¹⁵N tracer techniques and physical fractionation procedures was applied. Within 570 d after application of ¹⁵N labelled mustard litter to an agricultural cropland, the distribution of ¹⁵N was measured in particulate organic matter (POM) fractions and in fine mineral fractions (fine silt- and clay-sized fractions). After 570 d, only 2.5% of the initial ¹⁵N amount was found in POM fractions, with higher amounts in POM occluded in aggregates than in free POM. After this period, stabilization of the initial ¹⁵N in fine silt- and clay-sized fractions amounts to 10% in HY, but 20% in LY soils. 70% to 85% of the added ¹⁵N were lost. Initial decomposition of labelled material was faster in HY than in LY areas during the first year, but the remaining ¹⁵N amounts in POM fractions of the different areas were similar after 570 d. ¹⁵N amounts and concentrations in mineral-associated fractions increased within 160 d after application. From 160 to 570 d, HY and LY areas showed different ¹⁵N dynamics, resulting in a decline of ¹⁵N amounts in HY, but constant ¹⁵N amounts in LY soils. The results indicate faster decomposition processes in HY than in LY areas, due to different soil conditions, such as soil texture and water regime. The higher silt and clay contents of LY areas seem to promote N stabilization in fine mineral fractions. As a whole, N flows were higher in HY compared to LY areas, thus supporting higher yields and accelerated organic matter degradation due to higher N supply.     

Soil microbial dynamics in maize-growing soil under different tillage and residue management systems

The long-term impact of tillage and residue management on soil microorganisms was studied over the growing season in a sandy loam to loamy sand soil of southwestern Quebec, growing maize (Zea mays L.) monoculture. Tillage and residue treatments were first imposed on plots in fall 1991. Treatments consisted of no till, reduced tillage, and conventional tillage with crop residues either removed from (−R) or retained on (+R) experimental plots, laid out in a randomized complete block design. Soil microbial biomass carbon (SMB-C), soil microbial biomass nitrogen (SMB-N) and phospholipid fatty acid (PLFA) contents were measured four times, at two depths (0-10 and 10-20 cm), over the 2001 growing season. Sample times were: May 7 (preplanting), June 25, July 16, and September 29 (prior to corn harvest). The effect of time was of a greater magnitude than those attributed to tillage or residue treatments. While SMB-C showed little seasonal change (160 μg C g⁻¹ soil), SMB-N was responsive to post-emergence mineral nitrogen fertilization, and PLFA analysis showed an increase in fungi and total PLFA throughout the season. PLFA profiles showed better distinction between sampling time and depth, than between treatments. The effect of residue was more pronounced than that of tillage, with increased SMB-C and SMB-N (61 and 96%) in +R plots compared to −R plots. This study illustrated that measuring soil quality based on soil microbial components must take into account seasonal changes in soil physical and chemical conditions.     

Manipulating the N release from N labelled celery residues by using straw and vinasses

The aim of this laboratory study was to investigate the effect of straw and vinasses on the nitrogen (N) mineralization-immobilization turnover of celery residues during two periods (each simulating a time period from autumn till spring) under laboratory conditions. During the first period (1-198 d), ¹⁵N-labelled celery residues (1.1 g dry matter (DM) kg⁻¹ soil) were incubated together with straw (8.1 g DM kg⁻¹ soil), aiming to immobilize the N released from celery residues, followed by an incorporation of vinasses (1.9 g DM kg⁻¹ soil) after 84 d, with a view to remineralizing the immobilized celery-N. During the second period (198-380 d), the experimental set-up was repeated, except that non-labelled celery residues were used. Total N, mineral N and their ¹⁵N enrichments as well as microbial biomass N were determined at regular time intervals. During both periods, mixing celery residues with straw significantly increased microbial biomass N (90.5 and 40.5 mg N kg⁻¹ extra compared to celery only treatment) and decreased the amount of mineral N (reduction of 56.1 and 45.9 mg N kg⁻¹ soil compared to celery only treatment) and the celery-derived mineral ¹⁵N (0% of mineral celery-derived ¹⁵N in straw treatment compared to 35% of mineral celery-derived ¹⁵N in celery only treatment). After maximum immobilization, a natural remineralization (without addition of vinasses) of 32.2 (at day 198) and 11.1 mg N kg⁻¹ soil (at day 380) occurred in the straw treatment, but the mineral N content remained significantly lower than in the celery only treatment during the complete experiment, and the amount of remineralized celery-¹⁵N was very low (5.4% of celery-derived ¹⁵N after 380 d). Vinasses caused no real priming effect, although it did slightly increase the amount of remineralized celery-¹⁵N (+6.4% of celery-derived ¹⁵N at day 380 compared to the straw treatment), probably due an apparent added N interaction caused by displacement reactions with the soil microbial biomass.     

Short-term effects of dairy slurry amendment on carbon sequestration and enzyme activities in a temperate grassland

Land application of animal wastes from intensive grassland farming has resulted in growing environmental problems relating to greenhouse gas emissions, ammonia volatilisation, and nitrate and phosphorus leaching into surface and groundwater. We examined the short-term effects of dairy slurry amendment on carbon sequestration and enzyme activities in a temperate grassland (Southwest England). Slurry was collected from cows fed either on perennial ryegrass (C₃) or maize (C₄) silages. Fifty m³ ha⁻¹ of each of the obtained C₃ or C₄ slurries (δ¹³C=−30.7 and −21.3‰, respectively) were applied to a C₃ pasture soil with δ¹³C of −30.0±0.2‰. We found that water soluble organic carbon (WSOC) content was two to three times higher in the slurry amended plots compared with the unamended control. No significant change in the soil microbial biomass (SMB) carbon content was observed in the four weeks (772 h) following slurry application. Natural abundance ¹³C isotope analysis suggested a rapid initial incorporation (>25% within 2 h of application) of slurry-derived C in the SMB-C and WSOC pools of the 0-2 cm layer. Linear relationships were found between slurry-derived C in the whole soil, SMB, and WSOC for the 0-2 cm depth in the soil. Applied slurry-derived C was sequestered in the SMB pool in two phases. The first phase (0-48 h) was dominated by the incorporation of labile slurry C from the liquid phase, whereas beyond 48 h slurry-derived C was mainly from less mobile particulate C. No significant differences between treatments were found for invertase and xylanase. Urease activity was always higher in slurry treatments. Cellobiohydrolase, β-N-acetyl-glucosamidase, β-glucosidase and acid phosphatase activities became significantly higher in slurry treatments after 336 h. However, the observed temporal changes in enzyme activities were not correlated with the amounts of slurry-C incorporated in the SMB and WSOC pool.     

Atmospheric nitrate deposition and the microbial degradation of cellobiose and vanillin in a northern hardwood forest

Human activity has increased the amount of N entering terrestrial ecosystems from atmospheric NO₃⁻ deposition. High levels of inorganic N are known to suppress the expression of phenol oxidase, an important lignin-degrading enzyme produced by white-rot fungi. We hypothesized that chronic NO₃⁻ additions would decrease the flow of C through the heterotrophic soil food web by inhibiting phenol oxidase and the depolymerization of lignocellulose. This would likely reduce the availability of C from lignocellulose for metabolism by the microbial community. We tested this hypothesis in a mature northern hardwood forest in northern Michigan, which has received experimental atmospheric N deposition (30 kg NO₃⁻-N ha⁻¹ y⁻¹) for nine years. In a laboratory study, we amended soils with ¹³C-labeled vanillin, a monophenolic product of lignin depolymerization, and ¹³C-labeled cellobiose, a disaccharide product of cellulose degradation. We then traced the flow of ¹³C through the microbial community and into soil organic carbon (SOC), dissolved organic carbon (DOC), and microbial respiration. We simultaneously measured the activity of enzymes responsible for lignin (phenol oxidase and peroxidase) and cellobiose (β-glucosidase) degradation. Nitrogen deposition reduced phenol oxidase activity by 83% and peroxidase activity by 74% when compared to control soils. In addition, soil C increased by 76%, whereas microbial biomass decreased by 68% in NO₃⁻ amended soils. ¹³C cellobiose in bacterial or fungal PLFAs was unaffected by NO₃⁻ deposition; however, the incorporation of ¹³C vanillin in fungal PLFAs extracted from NO₃⁻ amended soil was 82% higher than in the control treatment. The recovery of ¹³C vanillin and ¹³C cellobiose in SOC, DOC, microbial biomass, and respiration was not different between control and NO₃⁻ amended treatments. Chronic NO₃⁻ deposition has stemmed the flow of C through the heterotrophic soil food web by inhibiting the activity of ligninolytic enzymes, but it increased the assimilation of vanillin into fungal PLFAs.     

华北平原典型农田土壤微生物生物量碳氮磷库的县域分布特征 ——以河北省曲周县为例

土壤微生物生物量是土壤中的活性养分库,直接参与土壤碳氮磷硫等元素的形态转化与生物地球化学循环过程,是反映土壤肥力与质量的重要生物指标.基于网格法采样,运用地统计学方法分析华北平原典型农田土壤微生物生物量碳氮磷库的空间分布特征及影响因子.结果表明:河北省曲周县域农田耕层(0～30 cm)土壤微生物生物量库在空间上呈斑块状...     

Dynamics of 13C‐labeled mustard litter (Sinapis alba) in particle‐size and aggregate fractions in an agricultural cropland with high‐ and low‐yield areas

The application of ¹³C‐labeled litter enables to study decomposition processes as well as the allocation of litter‐derived carbon to different soil C pools. ¹³Carbon‐labeled mustard litter was used in order to compare decomposition processes in an agricultural cropland with high‐yield (HY) and low‐yield (LY) areas, the latter being characterized by a finer texture and a lower organic‐C (OC) content. After tracer application, ¹³C concentrations were monitored in topsoil samples in particulate organic matter (POM) and in fine mineral fractions (silt‐ and clay‐sized fractions). After 568 d, approximately 5% and 10% of the initial ¹³C amount were found in POM fractions of LY and HY areas, respectively. Higher amounts were found in POM occluded in aggregates than in free POM. Medium‐term (0.5–2 y) storage of the initial ¹³C in fine silt‐ and clay‐sized fractions amounts to 10% in HY and LY soils, with faster enrichment but also faster disappearance of the ¹³C signal from LY soils. Amounts of 80%–90% of the added ¹³C were mineralized or leached in the observed period. Decomposition of free POM was faster in HY than in LY areas during the first year, but the remaining ¹³C amounts in occluded‐POM fractions were higher in HY soils after 568 d. High‐yield and low‐yield areas showed different ¹³C dynamics in fine mineral fractions. In LY soils, ¹³C amounts and concentrations in mineral‐associated fractions increased within 160 d after application and decreased in the following time period. In HY areas, a significant increase in ¹³C amounts did not occur until after 568 d. The results indicate initially faster decomposition processes in HY than in LY areas due to different soil conditions, such as soil texture and water regime. The higher silt and clay contents of LY areas seem to promote a faster aggregate formation and turnover, leading to a closer contact between POM and mineral surfaces in this area. This favors the OC storage in fine mineral fractions in the medium term. Lower aggregate formation and turnover in the coarser textured HY soil leads to a delayed C stabilization in silt‐ and clay‐sized fractions.     

Fungal biomass production and turnover in soil estimated using the acetate-in-ergosterol technique

We report the first attempt to estimate fungal biomass production in soil by correlating relative fungal growth rates (i.e., acetate incorporation into ergosterol) with fungal biomass increase (i.e., ergosterol) following amendments with dried alfalfa or barley straw in soil. The conversion factor obtained was then used in unamended soil, resulting in fungal biomass productions of 10-12 μg C g⁻¹ soil, yielding fungal turnover times between 130 and 150 days. Using a conversion factor from alfalfa-treated soil only resulted in two times higher estimates for biomass production and consequently lower turnover times. Comparing fungal biomass production with basal respiration indicated that these calculations overestimated the former. Still, the turnover times of fungal biomass in soil were in the same range as turnover times estimated in aquatic systems. The slow turnover of fungal biomass contrasts with the short turnover times found for bacteria. The study thus presents empirical data substantiating the theoretical division of bacteria and fungi into a fast and a slow energy channel, respectively, in the soil food web.     

C and N turnover in structurally intact soils of different texture

The turnover of native and applied C and N in undisturbed soil samples of different texture but similar mineralogical composition, origin and cropping history was evaluated at −10 kPa water potential. Cores of structurally intact soil with 108, 224 and 337 g clay kg⁻¹ were horizontially sliced and ¹⁵N-labelled sheep faeces was placed between the two halves of the intact core. The cores together with unamended treatments were incubated in the dark at 20 °C and the evolution of CO₂-C determined continuously for 177 d. Inorganic and microbial biomass N and ¹⁵N were determined periodically. Net nitrification was less in soil amended with faeces compared with unamended soil. When adjusted for the NO₃-N present in soil before faeces was applied, net nitrification became negative indicating that NO₃-N had been immobilized or denitrified. The soil most rich in clay nitrified least N and ¹⁵N. The amounts of N retained in the microbial biomass in unamended soils increased with clay content. A maximum of 13% of the faeces ¹⁵N was recovered in the microbial biomass in the amended soils. CO₂-C evolution increased with clay content in amended and unamended soils. CO₂-C evolution from the most sandy soil was reduced due to a low content of potentially mineralizable native soil C whereas the rate constant of C mineralization rate peaked in this soil. When the pool of potentially mineralizable native soil C was assumed proportional to volumetric water content, the three soils contained similar proportions of potentially mineralizable native soil C but the rate constant of C mineralization remained highest in the soil with least clay. Thus although a similar availability of water in the three soils was ensured by their identical matric potential, the actual volume of water seemed to determine the proportion of total C that was potentially mineralizable. The proportion of mineralizable C in the faeces was similar in the three soils (70% of total C), again with a higher rate constant of C mineralization in the soil with least clay. It is hypothesized that the pool of potentially mineralizable C and C rate constants fluctuate with the soil water content.     

Microbial use of maize cellulose and sugarcane sucrose monitored by changes in the C/C ratio

An arable soil with organic matter formed from C₃-vegetation was amended initially with maize cellulose (C₄-cellulose) and sugarcane sucrose (C₄-sucrose) in a 67-day laboratory incubation experiment with microcosms at 25 °C. The amount and isotopic composition (¹³C/¹²C) of soil organic C, CO₂ evolved, microbial biomass C, and microbial residue C were determined to prove whether the formation of microbial residues depends on the quality of the added C source adjusted with NH₄NO₃ to the same C/N ratio of 15. In a subsequent step, C₃-cellulose (3 mg C g⁻¹ soil) was added without N to soil to determine whether the microbial residues formed initially from C₄-substrate are preferentially decomposed to maintain the N-demand of the soil microbial community. At the end of the experiment, 23% of the two C₄-substrates added was left in the soil, while 3% and 4% of the added C₄-cellulose and C₄-sucrose, respectively, were found in the microbial biomass. The addition of the two C₄-substrates caused a significant 100% increase in C₃-derived CO₂ evolution during the 5-33 day incubation period. The addition of C₃-cellulose caused a significant 50% increase in C₄-derived CO₂ evolution during the 38-67 day incubation period. The decrease in microbial biomass C₄-C accounted for roughly 60% of this increase. Cellulose addition promoted microorganisms strongly able to recycle N immediately from their own tissue by “cryptic growth” instead of incorporating NO₃⁻ from the soil solution. The differences in quality of the microbial residues produced by C₄-cellulose and C₄-sucrose decomposing microorganisms are also reflected by the difference in the rates of CO₂ evolution, but not in the rates of net N mineralization.     

Dynamics of maize (Zea mays L.) leaf straw mineralization as affected by the presence of soil and the availability of nitrogen

An incubation experiment was carried out with maize (Zea mays L.) leaf straw to analyze the effects of mixing the residues with soil and N amendment on the decomposition process. In order to distinguish between soil effects and nitrogen effects for both the phyllospheric microorganisms already present on the surface of maize straw and soil microorganisms the N amendment was applied in two different placements: directly to the straw or to the soil. The experiment was performed in dynamic, automated microcosms for 22 days at 15 °C with 7 treatments: (1) untreated soil, (2) non-amended maize leaf straw without soil, (3) N amended maize leaf straw without soil, (4) soil mixed with maize leaf straw, (5) N amended soil, (6) N amended soil mixed with maize leaf straw, and (7) soil mixed with N amended maize leaf straw. ¹⁵NH₄¹⁵NO₃ (5 at%) was added. Gas emissions (CO₂, ¹³CO₂ and N₂O) were continuously recorded throughout the experiment. Microbial biomass C, biomass N, ergosterol, δ¹³C of soil organic C and of microbial biomass C as well as ¹⁵N in soil total N, mineral N and microbial biomass N were determined in soil samples at the end of the incubation. The CO₂ evolution rate showed a lag-phase of two days in the non-amended maize leaf straw treatment without soil, which was completely eliminated when mineral N was added. The addition of N generally increased the CO₂ evolution rate during the initial stages of maize leaf straw decomposition, but not the cumulative CO₂ production. The presence of soil caused roughly a 50% increase in cumulative CO₂ production within 22 days in the maize straw treatments due to a slower decrease of CO₂ evolution after the initial activity peak. Since there are no limitations of water or N, we suggest that soil provides a microbial community ensuring an effective succession of straw decomposing microorganisms. In the treatments where maize and soil was mixed, 75% of microbial biomass C was derived from maize. We concluded that this high contribution of maize using microbiota indicates a strong influence of organisms of phyllospheric origin to the microbial community in the soil after plant residues enter the soil.     

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.     

Response of microbial communities to water stress in irrigated and drought-prone tallgrass prairie soils

To better understand how water stress and availability affect the structure of microbial communities in soil, I measured the change in phospholipid fatty acids (PLFA) and the incorporation of ¹³C-labeled glucose into the PLFA following exposure to water stress. Overlaid on the laboratory water stress treatment, samples were collected from drought-prone and irrigated (11 years) tallgrass prairie soil (0-10 cm depth). In the laboratory, soils were either incubated at −250 kPa or dried steadily over a 3-d period to −45 MPa. On the fourth day, the dried samples were brought up to −250 kPa and then all samples received 250 μg of glucose-C (+4000 δ¹³C-PDB) solution that brought them to −33 kPa matric water potential. Samples were then extracted for PLFA following 6 and 24 h of incubation (25 °C). Non-metric multidimensional scaling (NMS) techniques and multi-response permutation procedure (MRPP) showed that the largest effect on the mol% distribution of PLFA was related to the field scale water addition experiment. In response to irrigation, the PLFA 16:1ω5, 18:1+, and 18:2ω6,9 showed increases, and a15:0, a17:0, and cy19:0 showed decreases in their respective mol%. Effects related to the induction of laboratory water stress were predominantly associated with a decrease in the mol% distribution of the putative fungal biomarker (18:2ω6,9) with little to no change in the mol% distribution of the bacterial biomarkers. Interestingly, the flow of C to the microbial community was not strongly related to any single PLFA, and differences were rather subtle, but multivariate MRPP detected change to the community structure related to the laboratory water stress treatment but not related to the 11 years of field irrigation. Our results suggest that both the total and the actively metabolizing bacterial community in soil were generally resistant to the effects of water stress brought by rewetting of dry soil. However, more research is needed to understand the nature of the fungal response to drying and rewetting in soil.     

Effects of fertilizer application and fire regime on soil microbial biomass carbon and nitrogen,and nitrogen mineralization in an Australian subalpine eucalypt forest

The effects of a range of fertilizer applications and of repeated low-intensity prescribed fires on microbial biomass C and N, and in situ N mineralization were studied in an acid soil under subalpine Eucalyptus pauciflora forest near Canberra, Australia. Fertilizer treatments (N, P, N+P, line + P, sucrose + P), and P in particular, tended to lower biomass N. The fertilizer effects were greatest in spring and smaller in summer and late actumn. Low-intensity prescribed fire lowered biomass N at a soil depth of 0–5 cm with the effect being greater in the most frequently burnt soils. No interactions between fire treatments, season, and depth were significant. Only the lime + P and N+P treatments significantly affected soil microbial biomass C contents. The N+P treatment increased biomass C only at 0–2.5 cm in depth, but the soil depth of entire 0–10 cm had much higher (>doubled) biomass C values in the line + P treatment. Frequent (two or three times a year) burning reduced microbial boomass C, but the reverse was true in soils under forest burn at intervals of 7 years. Soil N mineralization was increased by the addition of N and P (alone or in combination), line + P, and sucrose + P to the soil. The same was true for the ratio of N mineralization to biomass N. Soil N mineralization was retarded by repeated fire treatments, especially the more frequent fire treatment where rates were only about half those measured in unburnt soils. There was no relationship between microbial biomass N (kg N ha^-1) and the field rates of soil N mineralization (kg N ha^-1 month^-1). The results suggest that although soil microbial biomass N represents a distinct pool of N, it is not a useful measure of N turnover.     

Modification of the original chloroform fumigation extraction technique to allow measurement of δC of soil microbial biomass carbon

In studies of the soil microbial biomass C by the chloroform fumigation extraction (CFE) technique, biomass C is routinely extracted using 0.5 M K₂SO₄ solution. The excessive amounts of salts contained in the extracting solution pause a significant challenge in using ¹³C isotope techniques to study the nature of C in the soil microbial biomass. This is because the salts can affect the oxidation process and therefore hamper accurate mass spectromic analysis of dried extracts. In spite of this, no standard protocol exists for preparing the K₂SO₄ extracts for ¹³C isotope analysis. We have modified the original CFE method to allow measurement of the δ¹³C of soil microbial biomass C by using 2 M KCl instead of the usual 0.5 M K₂SO₄ solution to extract biomass C. Excess salts were removed by dialysis in 100 molecular weight cut off membranes, after which the extracts were freeze-dried and their δ¹³C measured using a mass spectrometer. The soil microbial biomass C and δ¹³C of 2 M KCl extracts were compared with those of 0.5 M K₂SO₄ extracts. There was excellent agreement between organic C and δ¹³C estimates for dialyzed 2 M KCl and 0.5 M K₂SO₄ extracts, but the speed of dialysis for the latter was very slow, making use of the former more rapid. These results suggest that in procedures where oxidation with potassium dichromate is not critical to analysis of soluble C, 2 M KCl may be used in place of 0.5 M K₂SO₄ to extract soil microbial biomass C for δ¹³C measurements. The new procedure is relatively easy and rapid for obtaining indices for both pool sizes and turnover rates of soil microbial biomass C and provides a promising approach to study soil organic C.     

Fate of gram-negative bacterial biomass in soil—mineralization and contribution to SOM

Soil microorganisms contribute to the formation of non-living soil organic matter (SOM) by metabolic transformation of plant-derived material. After cell death, their biomass components with a specific molecular character become incorporated into SOM imprinting its chemical properties, although this process has not yet been quantified. In order to elucidate the contribution to SOM formation, we investigated the fate of gram-negative bacterial model biomass (Escherichia coli usually introduced into soil with manure or feces) during incubation of soil with isotopically (¹³C) and genetically (lux gene) labeled cells. The decline of living cells was monitored by the loss of bioluminescence. The carbon turnover and mineralization was balanced by bulk soil stable isotope analysis, and the persistence of nucleic acids was investigated by PCR amplification of the lux gene. During incubation, the number of viable E. coli cells decreased rapidly (99.9% within the first 42 d) serving as substrate for other microorganisms or for the formation of SOM, and bioluminescent cells could only be detected during the first 56 d. However, the lux gene was still detected after 224 d, which indicates stabilization of DNA in SOM. Although the survival of E. coli in soil is limited, only about 65% of the added labeled biomass carbon was mineralized to ¹³CO₂ and 51% remained in soil after 224 d with an average ¹³C recovery of 117%. The amount of ¹³C found in the PLFA representative of living cells had decreased to 25% of the initial value, suggesting a proportional decrease of the ¹³C in the soil microbial biomass. The extent of this decrease is higher than the mineralization of the bulk E. coli C and thus the difference of around 25% has to be stabilized as metabolites, or in non-living SOM. The data provide evidence that the genetic information and a considerable part of the carbon from dying bacterial biomass were retained in both the soil microbial food web and in non-living SOM.     

Turnover of soil organic matter and of microbial biomass under C3-C4 vegetation change: Consideration of C fractionation and preferential substrate utilization

Two processes contribute to changes of the δ¹³C signature in soil pools: ¹³C fractionation per se and preferential microbial utilization of various substrates with different δ¹³C signature. These two processes were disentangled by simultaneously tracking δ¹³C in three pools - soil organic matter (SOM), microbial biomass, dissolved organic carbon (DOC) - and in CO₂ efflux during incubation of 1) soil after C₃-C₄ vegetation change, and 2) the reference C₃ soil.The study was done on the Ap horizon of a loamy Gleyic Cambisol developed under C₃ vegetation. Miscanthus giganteus - a perennial C₄ plant - was grown for 12 years, and the δ¹³C signature was used to distinguish between ‘old’ SOM (>12 years) and ‘recent’ Miscanthus-derived C (<12 years). The differences in δ¹³C signature of the three C pools and of CO₂ in the reference C₃ soil were less than 1‰, and only δ¹³C of microbial biomass was significantly different compared to other pools. Nontheless, the neglecting of isotopic fractionation can cause up to 10% of errors in calculations. In contrast to the reference soil, the δ¹³C of all pools in the soil after C₃-C₄ vegetation change was significantly different. Old C contributed only 20% to the microbial biomass but 60% to CO₂. This indicates that most of the old C was decomposed by microorganisms catabolically, without being utilized for growth. Based on δ¹³C changes in DOC, CO₂ and microbial biomass during 54 days of incubation in Miscanthus and reference soils, we concluded that the main process contributing to changes of the δ¹³C signature in soil pools was preferential utilization of recent versus old C (causing an up to 9.1‰ shift in δ¹³C values) and not ¹³C fractionation per se.Based on the δ¹³C changes in SOM, we showed that the estimated turnover time of old SOM increased by two years per year in 9 years after the vegetation change. The relative increase in the turnover rate of recent microbial C was 3 times faster than that of old C indicating preferential utilization of available recent C versus the old C.Combining long-term field observations with soil incubation reveals that the turnover time of C in microbial biomass was 200 times faster than in total SOM. Our study clearly showed that estimating the residence time of easily degradable microbial compounds and biomarkers should be done at time scales reflecting microbial turnover times (days) and not those of bulk SOM turnover (years and decades). This is necessary because the absence of C reutilization is a prerequisite for correct estimation of SOM turnover. We conclude that comparing the δ¹³C signature of linked pools helps calculate the relative turnover of old and recent pools.     

Combining field incubation with nitrogen-15 labelling to examine nitrogen transformations in low to high intensity grassland management systems

 The ¹⁵N isotope dilution method was combined with a field incubation technique to provide simultaneous measurements of gross and net rates of N turnover in three long-term swards: unfertilized (Z) or receiving N either from N fixation as clover (C), or as 200 kg fertilizer N ha^–1 year^–1 (F). Uniform N enrichment of soil microplots was achieved with a multi-point soil injector to measure mineralization/immobilization turnover and nitrification over a 4-day incubation. Net rates of mineralization ranged between 0.6 and 2.9 μg N g^–1 day^–1 and in all three treatments were approximately half the gross rates. Nitrification rates (gross) were between 1.0 and 1.6 μg N g^–1 day^–1. In the F treatment, the turnover of NH₄ ⁺-N and NO₃ ^–-N pools was on a 2- and 4-day cycle, respectively, whereas in the N-limited treatments (C and Z) turnover rates were faster, with the NO₃ ^–-N pools turning over twice as fast as the NH₄ ⁺-N pools. Therefore, available N was recycled more efficiently in the C and Z treatments, whereas in the F treatment a higher N pool size was maintained which would be more vulnerable to leakage. A large proportion of the added ¹⁵N was recovered in the soil microbial biomass (SMB), which represented a 4–5 times larger sink for N than the plant biomass. Although the C treatment had a significantly lower SMB than the grass-only treatments, there were no differences in microbial activity. Gross rates of nitrification increased along the gradient of N input intensity (i.e. Z<C<F), and the addition of a nitrification inhibitor (C₂H₂) tended to increase microbial immobilization, but did not influence plant N uptake. In this study, the value of combining different techniques to verify net rates was demonstrated and the improved methodology for ¹⁵N labelling of soil enabled measurements to be obtained from relatively undisturbed soil under natural field conditions. Received: 25 May 1999     

Microbial community composition and rhizodeposit-carbon assimilation in differently managed temperate grassland soils

Rhizodeposit-carbon provides a major energy source for microbial growth in the rhizosphere of grassland soils. However, little is known about the microbial communities that mediate the rhizosphere carbon dynamics, especially how their activity is influenced by changes in soil management. We combined a ¹³CO₂ pulse-labeling experiment with phospholipid fatty acid (PLFA) analysis in differently managed Belgian grasslands to identify the active rhizodeposit-C assimilating microbial communities in these grasslands and to evaluate their response to management practices. Experimental treatments consisted of three mineral N fertilization levels (0, 225 and 450 kg N ha⁻¹ y⁻¹) and two mowing frequencies (3 and 5 times y⁻¹). Phospholipid fatty acids were extracted from surface (0-5 cm) bulk (BU) and root-adhering (RA) soil samples prior to and 24 h after pulse-labeling and were analyzed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-c-IRMS). Soil habitats significantly differed in microbial community structure (as revealed by multivariate analysis of mol% biomarker PLFAs) as well as in gram-positive bacterial rhizodeposit-C uptake (as revealed by greater ¹³C-PLFA enrichment following pulse-labeling in RA compared to BU soil in the 450N/5M treatment). Mowing frequency did not significantly alter the relative abundance (mol%) or activity (¹³C enrichment) of microbial communities. In the non-fertilized treatment, the greatest ¹³C enrichment was seen in all fungal biomarker PLFAs (C16:1ω5, C18:1ω9, C18:2ω6,9 and C18:3ω3,6,9), which demonstrates a prominent contribution of fungi in the processing of new photosynthate-C in non-fertilized grassland soils. In all treatments, the lowest ¹³C enrichment was found in gram-positive bacterial and actinomycetes biomarker PLFAs. Fungal biomarker PLFAs had significantly lower ¹³C enrichment in the fertilized compared to non-fertilized treatments in BU soil (C16:1ω5, C18:1ω9) as well as RA soil (all fungal biomarkers). While these observations clearly indicated a negative effect of N fertilization on fungal assimilation of plant-derived C, the effect of N fertilization on fungal abundance could only be detected for the arbuscular mycorrhizal fungal (AMF) PLFA (C16:1ω5). On the other hand, increases in the relative abundance of gram-positive bacterial PLFAs with N fertilization were found without concomitant increases in ¹³C enrichment following pulse-labeling. We conclude that in situ¹³C pulse-labeling of PLFAs is an effective tool to detect functional changes of those microbial communities that are dominantly involved in the immediate processing of new rhizosphere-C.