Identification of Metabolically Active Rhizosphere Microorganisms by Stable Isotopic Probing of PLFA in Switchgrass

Root-derived rhizodeposits of recent photosynthetic carbon (C) are the foremost source of energy for microbial growth and development in rhizosphere soil. A substantial amount of photosynthesized C by the plants is translocated to belowground and is released as root exudates that influence the structure and function of soil microbial communities with potential inference in nutrient and C cycling in the ecosystem. We applied the ¹³C pulse chase labeling technique to evaluate the incorporation of rhizodeposit-C into the phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of switchgrass (Panicum virgatum L.). Soil samples of bulk and rhizosphere were taken at 1, 5, 10 and 20 days after labeling and analyzed for ¹³C enrichment in the microbial PLFAs. Temporal differences of ¹³C enrichment in PLFAs were more prominent than spatial differences. Among the microbial PLFA biomarkers, fungi and Gram-negative (GM-ve) bacterial PLFAs showed rapid enrichment with ¹³C compared to Gram-positive (GM+ve) and actinomycetes in rhizosphere soil. The ¹³C enrichment of actinomycetes biomarker PLFA significantly increased along with sampling time in both soils. PLFAs indicative to fungi, GM-ve and GM+ve showed a significant decrease in ¹³C enrichment over sampling time in the rhizosphere, but a decrease was also observed in GM-ve (16:1ω5c) and fungal biomarker PLFAs in the bulk soil. The relative ¹³C concentration in fungal PLFA decreased on day 10, whereas those of GM-ve increased on day 5 and GM+ve remained constant in the rhizosphere soil. However, the relative ¹³C concentrations of GM-ve and GM+ve increased on days 5 and 10, respectively, and those of fungal remain constant in the bulk soil. The present study demonstrates the usefulness of ¹³C pulse chase labeling together with PLFA analysis to evaluate the active involvement of microbial community groups for utilizing rhizodeposit-C.     

温度和秸秆质量调节与秸秆分解相关的微生物磷脂脂肪酸（PLFA）组成

Variations in temperature and moisture play an important role in soil organic matter (SOM) decomposition. However, relationships between changes in microbial community composition induced by increasing temperature and SOM decomposition are still unclear. The present study was conducted to investigate the effects of temperature and moisture levels on soil respiration and microbial communities involved in straw decomposition and elucidate the impact of microbial communities on straw mass loss. A 120-d litterbag experiment was conducted using wheat and maize straw at three levels of soil moisture (40%, 70%, and 90% of water-holding capacity) and temperature (15, 25, and 35°C). The microbial communities were then assessed by phospholipid fatty acid (PLFA) analysis. With the exception of fungal PLFAs in maize straw at day 120, the PLFAs indicative of Gram-negative bacteria and fungi decreased with increasing temperatures. Temperature and straw C/N ratio significantly affected the microbial PLFA composition at the early stage, while soil microbial biomass carbon (C) had a stronger effect than straw C/N ratio at the later stage. Soil moisture levels exhibited no significant effect on microbial PLFA composition. Total PLFAs significantly influenced straw mass loss at the early stage of decomposition, but not at the later stage. In addition, the ratio of Gram-negative and Gram-positive bacterial PLFAs was negatively correlated with the straw mass loss. These results indicated that shifts in microbial PLFA composition induced by temperature, straw quality, and microbial C sources could lead to changes in straw decomposition.     

Incorporation of C-labeled rice-straw-derived carbon into microbial communities in submerged rice field soil and percolating water

Rice straw is a major organic material applied to rice fields. The microorganisms growing on rice-straw-derived carbon have not been well studied. Here, we applied ¹³C-labeled rice straw to submerged rice soil microcosms and analyzed phospholipid fatty acids (PLFAs) in the soil and percolating water to trace the assimilation of rice-straw-derived carbon into microorganisms. PLFAs in the soil and water were markedly enriched with ¹³C during the first 3 days of incubation, which indicated immediate incorporation of rice-straw-derived carbon into microbial biomass. The enrichment of PLFAs in the percolating water with ¹³C suggested that microorganisms other than the population colonizing rice straw also assimilated rice-straw-derived carbon or that some bacterial groups were selectively released from the straw. The microbial populations could be categorized into two communities based on the carbon isotope data of the PLFAs: those derived from rice straw and those derived from soil organic matter (SOM). The composition of the PLFAs from the two communities differed, which indicated the assimilation of rice-straw-derived carbon by a subset of microbial populations. The composition of rice-straw-derived PLFAs in the percolating water was also distinct from that in the soil.     

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.     

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.     

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.     

Identification of groups of metabolically-active rhizosphere microorganisms by stable isotope probing of PLFAs

We combined microbial community phospholipid fatty acid (PLFA) analyses with an in situ stable isotope ¹³CO₂ labelling approach to identify microbial groups actively involved in assimilation of root-derived C in limed grassland soils. We hypothesized that the application of lime would stimulate more rapid ¹³C assimilation and turnover in microbial PLFAs. Four and 8 d after label application, 18:1ω9, 18:2ω6,9 (fungal biomarkers) and 16:1ω7, 18:1ω7, 19:0cy (Gram-negative bacterial biomarkers) showed the most ¹³C enrichment and rapid turnover rates. This suggests that these microorganisms were assimilating recently-photosynthesized root C inputs to soils. Contrary to our hypothesis, liming did not affect assimilation or turnover rates of ¹³C-labelled C. ¹³C stable isotope pulse-labelling technique paired with analyses of PLFA microbial biomarkers shows promise for in situ investigations of microbial function in soils.     

Linking Microbial Community Dynamics Associated with Rhizosphere Carbon Flow in a Biofuel Crop (Jatropha curcas L.)

Photosynthetically derived rhizodeposits are an important source of carbon (C) for microbes in root vicinity and can influence the microbial community dynamics. Pulse labeling of carbon dioxide (¹³CO₂) coupled with stable isotope probing techniques have potential to track recently fixed photosynthate into rhizosphere microbial taxa. Therefore, the present investigation assessed the microbial community change associated with the rhizosphere and bulk soil in Jatropha curcas L. (a biofuel crop) by combining phospholipid fatty acid (¹³C-PLFA) profiling using a stable isotope ¹³CO₂ labeling approach. The labeling (¹³C) took place after 45 days of germination, PLFAs were extracted from both soils (rhizosphere and bulk) after 1 and 20 days pulse labeling and analyzed by gas chromatography-isotope ratio mass spectrometry. There was no significant temporal effect on the PLFA profiles in the bulk soil, but significantly increased abundance of Gram positive (i15:0) and Gram negative (16:1ω7c and 16:1ω5c) biomarkers was observed in the rhizosphere soil from day 1 to day 20 after labeling. The Gram negative (16:1ω7c) decreased and fungal (18:2ω6,9c) increased significantly in rhizospheric soil compared to bulk soil after day 1 of labeling. Whereas, after 20 days of labeling, the Gram negative biomarker (16:1ω7c and 18:1ω7c) decreased and Gram positive (a15:0) increased significantly in rhizospheric soil compared to bulk soil. One day following labeling, i15:0, a15:0, i16:0, 16:1ω5c, 16:0, i17:0, a17:0, 18:2ω6,9c, 18:1ω9c, and 18:0 PLFAs were significantly more enriched in δ¹³C in the rhizosphere than in the bulk soil. Twenty days after labeling, 16:1ω5c (Gram negative) and 18:2ω6,9c (fungal) were significantly more enriched in δ¹³C in the rhizosphere than in the bulk soil. These results shows the effectives of PLFA coupled using the pulse chase labeling technique to examine the microbial community changes in response to recently fixed photosynthetic C flow in rhizodeposits.     

Linking microbial community structure and allocation of plant-derived carbon in an organic agricultural soil using 13CO2 pulse-chase labelling combined with 13C-PLFA profiling

We conducted a ¹³CO₂ pulse-chase labelling experiment in a drained boreal organic (peat) soil cultivated with perennial crop, reed canary grass (RCG; Phalaris arundinacea) to study the flow of carbon from plants to soil microbes. Both limed and unlimed soils were studied, since liming is a common agricultural practice for acidic organic soils. Soil samples taken within three months after the labelling and three times in the following year were used for the δ¹³C analysis of microbial phospholipid fatty acids (PLFAs), root sugars and root lipids. We estimated the contribution of carbon from root exudates to microbial PLFA synthesis. The flow of carbon from plants to microbes was fast as the label allocation in PLFAs had a peak 1–3 days after labelling. The results showed that fungi were important in the incorporation of fresh, plant-derived carbon, including root sugars. None of the main microbial PLFA biomarker groups (fungi, Gram-positive bacteria, Gram-negative bacteria, arbuscular mycorrhizal fungi) was completely lacking label over the measurement period. One year after the labelling, when the labelled carbon was widely distributed into plant biomass and soil, bacterial biomarkers increased their share of the label allocation. Liming had a minor effect on the label allocation rate into PLFAs. The mixing model approach used to calculate the root exudate contribution to microbial biomass resulted in a highly conservative estimate of utilization of this important C-source (0–6.5%, with highest incorporation into fungi). In summary, the results of this study provide new information about the role of various microbial groups in the turnover of plant-derived, fresh carbon in boreal organic soil.     

Incorporation of 13C-labelled rice rhizodeposition carbon into soil microbial communities under different water status

The overall processes by which carbon is fixed by plants in photosynthesis then released into the soil by rhizodeposition and subsequently utilized by soil micro-organisms, links the atmospheric and soil carbon pools. The objective of this study was to determine the plant derived ¹³C incorporated into the phospholipid fatty acid (PLFA) pattern in paddy soil, to test whether utilization of rice rhizodeposition carbon by soil micro-organisms is affected by soil water status. This is essential to understand the importance of flooded conditions in regulating soil microbial community structure and activity in wetland rice systems. Rice plants were grown in soil derived from a paddy system under controlled irrigation (CI), or with continuous waterlogging (CW). Most of the ¹³C-labelled rice rhizodeposition carbon was distributed into the PLFAs 16:0, 18:1ω7 and 18:1ω9 in both the CW and CI treatments. The bacterial PLFAs i15:0 and a15:0, both indicative of gram positive bacteria, were relatively more abundant in the treatments without rice plants. When rice plants were present rates of ¹³C-incorporation into i15:0 and a15:0 was slow; the microbes containing these PLFAs may derive most of their carbon from more recalcitrant C (soil organic matter). PLFAs, 18:1ω7 and 16:1ω7c, indicative of gram negative bacteria showed a greater amount incorporation of labelled plant derived carbon in the CW treatment. In contrast, 18:2ω6,9 indicative of fungi and 18:1ω9 indicative of aerobes but also potentially fungi and plant roots had greater incorporation in the CI treatment. The greater root mass concomitant with lower incorporation of ¹³C into the total PLFA pool in the CW treatment suggests that the microbial communities in wetland rice soil are limited by factors other than substrate availability in flooded conditions. In this study differing soil microbial communities were established through manipulating the water status of paddy soils. Steady state ¹³C labelling enabled us to determine that the microbial community utilizing plant derived carbon was also affected by water status.     

Microscale distribution and function of soil microorganisms in the interface between rhizosphere and detritusphere

The rhizosphere and the detritusphere are hot spots of microbial activity, but little is known about the interface between rhizosphere and detritusphere. We used a three-compartment pot design to study microbial community structure and enzyme activity in this interface. All three compartments were filled with soil from a long-term field trial. The two outer compartments were planted with maize (root compartment) or amended with mature wheat shoot residues from a free air CO₂ enrichment experiment (residue compartment) and were separated by a 50 μm mesh from the inner compartment. Soil, residues and maize differed in ¹³C signature (δ¹³C soil −26.5‰, maize roots −14.1‰ and wheat residues −44.1‰) which allowed tracking of root- and residue-derived C into microbial phospholipid fatty acids (PLFA). The abundance of bacterial and fungal PLFAs showed clear gradients with highest abundance in the first 1–2 mm of the root and residue compartment, and generally higher values in the vicinity of the residue compartment. The δ¹³C of the PLFAs indicated that soil microorganisms incorporated more carbon from the residues than from the rhizodeposits and that the microbial use of wheat residue carbon was restricted to 1 mm from the residue compartment. Carbon incorporation into soil microorganisms in the interface was accompanied by strong microbial N immobilisation evident from the depletion of inorganic N in the rhizosphere and detritusphere. Extracellular enzyme activities involved in the degradation of organic C, N and P compounds (β-glucosidase, xylosidase, acid phosphatase and leucin peptidase) did not show distinct gradients in rhizosphere or detritusphere. Our microscale study showed that rhizosphere and detritusphere differentially influenced microbial C cycling and that the zone of influence depended on the parameter assessed. These results are highly relevant for defining the size of different microbial hot spots and understanding microbial ecology in soils.     

Microbial community composition and carbon cycling within soil microenvironments of conventional, low-input, and organic cropping systems

This study coupled stable isotope probing with phospholipid fatty acid analysis (¹³C-PLFA) to describe the role of microbial community composition in the short-term processing (i.e., C incorporation into microbial biomass and/or deposition or respiration of C) of root- versus residue-C and, ultimately, in long-term C sequestration in conventional (annual synthetic fertilizer applications), low-input (synthetic fertilizer and cover crop applied in alternating years), and organic (annual composted manure and cover crop additions) maize-tomato (Zea mays - Lycopersicum esculentum) cropping systems. During the maize growing season, we traced ¹³C-labeled hairy vetch (Vicia dasycarpa) roots and residues into PLFAs extracted from soil microaggregates (53-250 μm) and silt-and-clay (<53 μm) particles. Total PLFA biomass was greatest in the organic (41.4 nmol g⁻¹ soil) and similar between the conventional and low-input systems (31.0 and 30.1 nmol g⁻¹ soil, respectively), with Gram-positive bacterial PLFA dominating the microbial communities in all systems. Although total PLFA-C derived from roots was over four times greater than from residues, relative distributions (mol%) of root- and residue-derived C into the microbial communities were not different among the three cropping systems. Additionally, neither the PLFA profiles nor the amount of root- and residue-C incorporation into the PLFAs of the microaggregates were consistently different when compared with the silt-and-clay particles. More fungal PLFA-C was measured, however, in microaggregates compared with silt-and-clay. The lack of differences between the mol% within the microbial communities of the cropping systems and between the PLFA-C in the microaggregates and the silt-and-clay may have been due to (i) insufficient differences in quality between roots and residues and/or (ii) the high N availability in these N-fertilized cropping systems that augmented the abilities of the microbial communities to process a wide range of substrate qualities. The main implications of this study are that (i) the greater short-term microbial processing of root- than residue-C can be a mechanistic explanation for the higher relative retention of root- over residue-C, but microbial community composition did not influence long-term C sequestration trends in the three cropping systems and (ii) in spite of the similarity between the microbial community profiles of the microaggregates and the silt-and-clay, more C was processed in the microaggregates by fungi, suggesting that the microaggregate is a relatively unique microenvironment for fungal activity.     

Degradation and humification of maize straw in soil microcosms inoculated with simple and complex microbial communities

Microbial communities are responsible for soil organic matter cycling and thus for maintaining soil fertility. A typical Orthic Luvisol was freed from organic carbon by thermal destruction at 600°C. Then the degradation and humification of ¹⁴C‐labelled maize straw by defined microbial communities was analysed. To study the role of microbial diversity on the humification of plant material, microcosms containing sterilized soil were inoculated with a natural microbial community or with microbial consortia consisting of bacterial and fungal soil isolates. Within 6 weeks, 41 ± 4% of applied ¹⁴C‐labelled maize straw was mineralized in the soil microcosms containing complex communities derived from a soil suspension, whilst the most efficient communities composed of soil isolates mineralized less than 35%. The humification products were analysed by solution state ¹³C‐NMR‐spectroscopy and gel permeation chromatography (GPC). The analyses of humic acids extracts by solution state ¹³C‐NMR‐spectroscopy revealed no difference in the development of typical chemical functional groups for humic substances during incubation. However, the increase in specific molecular size fractions of the extracted humic acids occurred only after inoculation with complex communities, but not with defined isolates. While it seems to be true that redundancy in soil microbial communities contributes to the resilience of soils, specific soil functions may no longer be performed if a microbial community is harshly affected in its diversity or growth conditions.     

Decomposition of maize residues after manipulation of colonization and its contribution to the soil microbial biomass

A 28-day incubation experiment at 12°C was carried out on the decomposition of maize leaf litter to answer the questions: (1) Is the decomposition process altered by chemical manipulations due to differences in the colonization of maize leaf litter? (2) Do organisms using this maize material contribute significantly to the soil microbial biomass? The extraction of the maize straw reduced its initial microbial biomass C content by 25%. Fumigation and extraction eliminated the microbial biomass by 88%. In total, 17% of added maize straw C was mineralized to CO₂ during the 28-day incubation at 12°C in the treatment with non-manipulated straw. Only 14% of added C was mineralized in the treatment with extracted straw as well as in the treatment with fumigated and extracted straw. The net increase in microbial biomass C was 79 μg g^?1 soil in the treatment with non-manipulated straw and an insignificant 9 μg g^?1 soil in the two treatments with manipulated straw. However, the net increase did not reflect the fact that the addition of maize straw replaced an identical 58% (≈180 μg g^?1 soil) of the autochthonous microbial biomass C₃-C in all three straw treatments. In the two treatments with manipulated straw, the formation of maize-derived microbial biomass C₄-C was significantly reduced by 25%. In the three straw treatments, the ratio of fungal ergosterol-to-microbial biomass C ratio showed a constant 60% increase compared to the control, and the contents of glucosamine and muramic acid increased by 18%. The average fungal C/bacterial C ratio was 3.6 in the soil and 5.0 in the recovered maize straw, indicating that fungal dominance was not altered by the initial chemical manipulations of the maize straw-colonizing microorganisms.     

Microbial phospholipid biomarkers and stable isotope methods help reveal soil functions

This review targets microbial phospholipid biomarkers, their isotope analysis and their ability to reveal soil functions. The amount and composition of phospholipid fatty acids (PLFAs) measured in environmental samples strongly depend on the methodology. To achieve comparable results the extraction, separation and methylation method must be kept constant. PLFAs patterns are sensitive to microbial community shifts even though the taxonomic resolution of PLFAs is low. The possibility to easily link lipid biomarkers with stable isotope techniques is identified as a major advantage when addressing soil functions. Measurement of PLFA isotopic ratios is sensitive and enables detecting isotopic fractionation. The difference between the carbon isotopic ratio of single PLFAs and their substrate (Δ¹³C) can vary between −6 and +11‰. This difference derives from the fractionation during biosynthesis and from substrate inhomogeneity. Consequently, natural abundance studies are restricted to quantifying substrate uptake of the total microbial biomass. In contrast, artificial labelling enables quantifying carbon uptake into single PLFAs, but labelling success depends on homogeneous and undisturbed label application. Current developments in microbial ecology (e.g. ¹³C and ¹⁵N proteomics) and isotope techniques (online monitoring of CO₂ isotope ratios) will likely improve soil functional interpretations in the future. ¹³C PLFA analysis will continue to contribute because it is affordable, sensitive and allows frequent sampling combined with the use of small amounts of ¹³C label.     

Tracing the source and fate of dissolved organic matter in soil after incorporation of a C labelled residue: A batch incubation study

While dissolved organic matter (DOM) in soil solution is a small but reactive fraction of soil organic matter, its source and dynamics are unclear. A laboratory incubation experiment was set up with an agricultural topsoil amended with ¹³C labelled maize straw. The dissolved organic carbon (DOC) concentration in soil solution increased sharply from 25 to 186 mg C L⁻¹ 4 h after maize amendment, but rapidly decreased to 42 mg C L⁻¹ and reached control values at and beyond 2 months. About 65% of DOM was straw derived after 4 h, decreasing to 29% after one day and only 1.3% after 240 days. A significant priming effect of the straw on the release of autochthonous DOM was found. The DOM fractionation with DAX-8 resin revealed that 98% of the straw derived DOM was hydrophilic in the initial pulse while this hydrophilic fraction was 20-30% in control samples. This was in line with the specific UV absorbance of the DOM which was significantly lower in the samples amended with maize residues than in the control samples. The δ¹³C of the respired CO₂ matched that of DOC in the first day after amendment but exceeded it in following days. The straw derived C fractions in respired CO₂ and in microbial biomass were similar between 57 and 240 days after amendment but were 3-10 fold above those in the DOM. This suggests that the solubilisation of C from the straw is in steady state with the DOM degradation or that part of the straw is directly mineralised without going into solution. This study shows that residue application releases a pulse of hydrophilic DOM that temporarily (<3 days) dominates the soil DOM pool and the degradable C. However, beyond that pulse the majority of DOM is derived from soil organic matter and its isotope signature differs from microbial biomass and respired C, casting doubt that the DOM pool in the soil solution is the major bioaccessible C pool in soil.     

Soil microbial community dynamics and phenanthrene degradation as affected by rape oil application

The effects of rape oil application on soil microbial communities and phenanthrene degradation were characterized by examining phenanthrene concentrations, changes in microbial composition and incorporation of [¹³C] phenanthrene-derived carbon into phospholipid fatty acids (PLFAs). A Haplic Chernozem was incubated with and without rape oil in combination with and without phenanthrene over 60 days. High-performance liquid chromatography (HPLC) analysis showed a net reduction in extractable phenanthrene in the soils treated with rape oil but no net reduction in the soils without rape oil. Rape oil application increased the total PLFA content and changed microbial community composition predominantly due to growth of fungal groups and Gram-positive bacterial groups. Under rape oil and phenanthrene amendment all detected microbial groups grew until day 24 of incubation. The ¹³C PLFA profiles showed ¹³C enrichment for the PLFAs i14:0, 15:0, 18:0, 18:1ω5 and the fungal biomarker 18:2ω6,9 under rape oil application. Fungal PLFA growth was highest among detected all PLFAs, but its ¹³C incorporation was lower compared to the Gram-positive and Gram-negative bacteria PLFAs. Our results demonstrate the effect of rape oil application on the abundance of microbial groups in soil treated with phenanthrene and its impact on phenanthrene degradation.     

甜玉米/白三叶草秸秆还田的碳氮矿化研究

豆科/禾本科作物间套作后进行秸秆还田能补充土壤养分,缓解集约化农业生产对环境的压力.根据田间甜玉米/白三叶草套种各作物的秸秆产量,在恒温恒湿条件下进行室内培养,探讨秸秆不同方式还田后土壤微生物量碳、微生物量氮、呼吸产生的CO2和矿化产生的无机氮的变化规律.研究发现,各施肥处理的土壤微生物量碳、微生物量氮均在培养前期出现峰值,后期平稳降低;甜玉米秸秆和白三叶草绿肥同时还田的土壤微生物量碳、微生物量氮在各培养时期均最大,峰值分别达529.57 mg·kg-1和75.50 mg·kg-1,土壤呼吸产生的CO2最多;白三叶草绿肥单独还田有利于土壤无机氮的释放,培养第26 d 无机氮达到最大值,为29.81 mg·kg-1,之后一直在对照处理的1.60倍以上,第80 d达到2.48倍;甜玉米秸秆单独还田不利于土壤无机氮的释放,培养的第26 d至结束,甜玉米秸秆处理的无机氮为对照的13%～53%,最大为7.51 mg·kg-1;甜玉米秸秆配施尿素,短期内不利于土壤无机氮矿化.结果表明,施用有机物料能引起土壤有机质的短期快速转化,甜玉米秸秆和白三叶草绿肥配施有利于维持较大基数的土壤微生物量,单施白三叶草绿肥土壤微生物活性强,最有利于土壤速效氮的释放.     

水分含量对秸秆还田土壤碳矿化和微生物特性的影响

An 80-d incubation experiment was conducted to investigate straw decomposition,the priming effect and microbial characteristics in a non-fertilized soil(soil 1) and a long-term organic manure-fertilized soil(soil 2) with and without13 C-labeled maize straw amendment under different moisture levels. The soil 2 showed a markedly higher priming effect,microbial biomass C(Cmic),and β-glucosidase activity,and more abundant populations of bacteria and fungi than the soil 1. Also,soil CO2 emission,Cmic,β-glucosidase activity,and bacterial and fungal population sizes were substantially enhanced by straw amendment. In the presence of straw,the amount of straw mineralization and assimilation by microbes in the soil at 55% of water holding capacity(WHC) were significantly higher by 31% and 17%,respectively,compared to those at 25% of WHC. In contrast,β-glucosidase activity and fungal population size were both enhanced as the moisture content decreased. Cmicdecreased as straw availability decreased,which was mainly attributed to the reduction of straw-derived Cmic. Amended soils,except the amended soil 2 at 25% of WHC,had a more abundant fungal population as straw availability decreased,indicating that fungal decomposability of added straw was independent of straw availability. Non-metric multidimensional scaling analysis based on fungal denatured gradient gel electrophoresis band patterns showed that shifts in the fungal community structure occurred as water and straw availability varied. The results indirectly suggest that soil fungi are able to adjust their degradation activity to water and straw availability by regulating their community structure.     

Nitrogen dynamics and microbial response in soil amended with either olive pulp or its by-products after biogas production

Olive pulp (OP), the residual material of a two-phase olive oil extraction system, and effluents from hydrogen (EH₂) and methane (ECH₄) production, have been evaluated as soil amendments particularly for their impact on soil mineral nitrogen (N) dynamics, gross N mineralization, and soil microbial biomass N (N_mic). Both N transformation and microbial growth were mainly influenced by the amount and quality of added organic carbon (C). Both OP and EH₂, which contain more carbohydrates and lipids than polyphenolic compounds, stimulated NO₃ ⁻ immobilization during the early incubation period and increased N_mic, saprophytic fungi, and N mineralization. On the contrary, soil amended with ECH₄, which is characterized by the lowest C content but the highest content of polyphenolic compounds, behaved as the control; neither NO₃ ⁻ immobilization nor microbial growth were observed and gross N mineralization was stimulated only at the beginning of the incubation period. Bacterial plate count was significantly correlated with direct bacterial count and fungal count was correlated with N_mic. Therefore, it is suggested that both bacterial and fungal plate counts may be used as indicators of the overall bacterial and fungal populations inhabiting soil, respectively. The knowledge of the impact of these materials on soil N dynamics is crucial for their correct use in agriculture because it has been shown that NO₃ ⁻ availability can be strongly influenced by the addition of different amounts and quality of organic amendment.