Microbial community utilization of added carbon substrates in response to long-term carbon input manipulation

The chemical composition and quantity of plant inputs to soil are primary factors controlling the size and structure of the soil microbial community. Little is known about how changes in the composition of the soil microbial community affect decomposition rates and other ecosystem functions. This study examined the degradation of universally ¹³C-labeled glucose, glutamate, oxalate, and phenol in soil from an old-growth Douglas-fir (Pseudotsuga menziesii)—western hemlock (Tsuga heterophylla) forest in the Oregon Cascades that has experienced 7 y of chronic C input manipulation. The soils used in this experiment were part of a larger Detritus Input and Removal Treatment experiment and have received normal C inputs (control), doubled wood inputs, or root and litter input exclusion (no inputs). Soil from the doubled wood treatment had a higher fungal:bacterial ratio, and soil from the no inputs treatment had a lower fungal:bacterial ratio, than the control soil. Differences in the utilization of the compounds added to the field-manipulated soils were assessed by following the ¹³C tracer into microbial biomass and respiration. In addition, ¹³C-phospholipid fatty acids (PLFA) analysis was used to examine differential microbial utilization of the added substrates. Glucose and glutamate were metabolized similarly in soils of all three litter treatments. In contrast, the microbial community in the double wood soil respired more added phenol and oxalate, whereas microbes in the no inputs soil respired less added phenol and oxalate, than the control soil. Phenol was incorporated primarily into fungal PLFA, especially in soil of the double wood treatment. The addition of all four substrates led to enhanced degradation of soil organic matter (priming) in soils of all three litter treatments, and was greater following the addition of phenol and oxalate as compared to glucose and glutamate. Priming was greater in the no inputs soil as compared to the control or doubled wood soils. These results demonstrate that altering plant inputs to soil can lead to changes in microbial utilization of C compounds. It appears that many of these changes are the result of alteration in the size and composition of the microbial community.     

Do same-aged but different height Norway spruce (Picea abies) clones affect soil microbial community?

The influence of individual trees in monocrop forests on soil microbial communities is poorly understood. We measured basal respiration, substrate-induced respiration and phospholipid fatty acids (PLFA), bacterial growth rate with the ³H-thymidine incorporation technique and fungal growth rate as ¹⁴C-acetate incorporation into ergosterol to investigate whether slow- and fast-growing 12-year-old Norway spruce (Picea abies) clones have affected differently on their associated soil microbial communities. Understorey vegetation, soil chemical properties and elemental concentrations of needles were also determined. The slow- and fast-growing spruce clones differed in PLFA profiles, understorey vegetation and elemental concentrations in needles suggesting that spruce clones have directly or indirectly affected soil microbes.     

Carbon flow from C-labeled straw and root residues into the phospholipid fatty acids of a soil microbial community under field conditions

To better understand how residue quality and seasonal conditions influence the flow of C from both root and straw residues into the soil microbial community, we followed the incorporation of ¹³C-labeled crimson clover (Trifolium incarnatum) and ryegrass (Lolium multiflorum) root and straw residues into the phospholipid fatty acids (PLFA) of soil microbial biomass. After residue incorporation under field conditions in late summer (September), the ¹³C content of soil PLFA was measured in September, October, and November, 2002, and April and June, 2003. Multivariate non-metric multidimensional scaling techniques showed that the distribution of ¹³C among microbial PLFA differed among the four primary treatments (ryegrass straw and roots, clover straw and roots). Regardless of treatment, some PLFA remained poorly labeled with ¹³C throughout much of the study (16:1ω5, 10Me17:0; 0-5%), whereas other PLFA consistently contained a larger percentage of residue-derived C (16:0; 18:1ω9, 18:2ω6,9; 10-25%). The distribution of residue ¹³C among individual PLFA differed from the relative contributions of individual PLFA (mol%) to total PLFA-C, suggesting that a subset of the soil biomass was primarily responsible for assimilating residue-derived C. The distribution of ¹³C among soil PLFA differed between the sampling times, indicating that residue properties and soil conditions influenced which members of the community were assimilating residue-derived C. Our findings will provide the foundation for further studies to identify the nature of the community members responsible for residue decomposition at different times of the year, and what factors account for the dynamics of the community involved.     

Carbon flow from C-labeled clover and ryegrass residues into a residue-associated microbial community under field conditions

Microbial colonization of soil-incorporated, ¹³C-labeled, crimson clover and ryegrass straw residues was followed under western Oregon field conditions from late summer (September) to the following early summer (mid-June) by measuring the ¹³C content of phospholipid fatty acid (PLFA) extracted from residues recovered from soil. Residue type influenced the rate of appearance of specific PLFA during early decomposition, with branch chain bacterial PLFA (i15:0, a15:0, i16:0) appearing on clover and ryegrass residues in October and November, respectively. By April, additional PLFA (16:1ω5, 16:1ω7, cy17:0, 18:0, 18:1ω9) had appeared on both residues. Between April and June, microbial community structure shifted again with significant increases (cy17:0, 18:0, 18:1ω9), and decreases (18:1ω7+10Me18:0) detected in the quantities of specific PLFA on both residue types. In the case of clover, the PLFA-C was derived primarily from residue C (85-100%), whereas in the case of ryegrass, both residue C (57-66%), and soil C contributed substantially to the PLFA-C.     

The microbial community composition changes rapidly in the early stages of decomposition of wheat residue

It is generally accepted that during the early stages of residue decomposition, easily available compounds are decomposed, leading to a relative increase in more recalcitrant compounds in the later stages of decomposition and that these changes in substrate availability are associated with changes in microbial community composition. However most studies on residue decomposition are conducted over several weeks or months; little is known about the changes in microbial community composition in the first weeks of decomposition. To address this knowledge gap, we incubated wheat residues inoculated with a microbial suspension in mesh bags buried in sand for 30 days, with sampling on days 0, 2, 4, 6, 8, 10, 15, 20, 25 and 30. Of the C added with the residues, 10, 18 and 25% had been respired on days 10, 20 and 30, respectively. The sum of PLFAs (phospholipid fatty acids), as an indicator of microbial biomass, increased strongly in the first 4 days and then decreased. The concentration of bacterial fatty acids was maximal on days 2 and 4, whereas the concentration of fungal fatty acids peaked on day 15. Microbial community composition (based on PLFA patterns) changed rapidly, with significant changes in the first 8 days and from day 8 to day 20. There were no significant changes in microbial community composition after day 20. The concentration of water-soluble C decreased strongly in the first 8 days, suggesting that the rapid changes in microbial community in this period are related to the changes in water-soluble C. Residue C chemistry, assessed by ¹³C NMR spectroscopy, changed little during the incubation period. This study showed that microbial community composition in decomposing residues changes rapidly in the first 1-2 weeks, which is, at least partly, the result of competition for the easily available compounds in the water-soluble fraction. After depletion of the water-soluble compounds, the microbial community composition changes more slowly.     

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.     

Does biochar interfere with standard methods for determining soil microbial biomass and phenotypic community structure?

Due to its high sorption affinity for organic compounds, biochar may interfere with extraction procedures involving such compounds used for microbially-related assays commonly applied to soils. Here we assessed the impact of two biochars (derived from pine bark and produced at 300 and 600 °C) at three concentrations (0, 12.5, and 50 g kg⁻¹) in three distinct arable soils with contrasting textural classes (loamy sand, sandy loam, and clay) on the determination of soil microbial biomass C by fumigation–extraction, fungal biomass by ergosterol analysis, and microbial community structure as defined by phospholipid fatty acid (PLFA) profiling. Biochar did not affect the apparent concentration of soil microbial biomass C and had no significant impact on apparent PLFA profiles. By contrast, the apparent extraction efficiency of ergosterol was affected dependent on soil type, biochar production temperature, and biochar concentration. Nonetheless, ergosterol contents of biochar-amended soils can be accurately estimated by correcting for reduced recovery using an ergosterol spike.     

Microbial community abundance and structure are determinants of soil organic matter mineralisation in the presence of labile carbon

Altered rates of native soil organic matter (SOM) mineralisation in the presence of labile C substrate (‘priming’), is increasingly recognised as central to the coupling of plant and soil-biological productivity and potentially as a key process mediating the C-balance of soils. However, the mechanisms and controls of SOM-priming are not well understood. In this study we manipulated microbial biomass size and composition (chloroform fumigation) and mineral nutrient availability to investigate controls of SOM-priming. Effects of applied substrate (¹³C-glucose) on mineralisation of native SOM were quantified by isotopic partitioning of soil respiration. In addition, the respective contributions of SOM-C and substrate-derived C to microbial biomass carbon (MBC) were quantified to account for pool-substitution effects (‘apparent priming’). Phospholipid fatty acid (PLFA) profiles of the soils were determined to establish treatment effects on microbial community structure, while the ¹³C-enrichment of PLFA biomarkers was used to establish pathways of substrate-derived C-flux through the microbial communities. The results indicated that glucose additions increased SOM-mineralisation in all treatments (positive priming). The magnitude of priming was reduced in fumigated soils, concurrent with reduced substrate-derived C-flux through putative SOM-mineralising organisms (fungi and actinomycetes). Nutrient additions reduced the magnitude of positive priming in non-fumigated soils, but did not affect the distribution of substrate-derived C in microbial communities. The results support the view that microbial community composition is a determinant of SOM-mineralisation, with evidence that utilisation of labile substrate by fungal and actinomycete (but not Gram-negative) populations promotes positive SOM-priming.     

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 dynamics of soil microbial phospholipid fatty acids to carbon mineralization in a C natural abundance experiment: Impact of heavy metals and acid rain

A ¹³C natural abundance experiment including GC-c-IRMS analysis of phospholipid fatty acids (PLFAs) was conducted to assess the temporal dynamics of the soil microbial community and carbon incorporation during the mineralization of plant residues under the impact of heavy metals and acid rain. Maize straw was incorporated into (i) control soil, (ii) soil irrigated with acid rain, (iii) soil amended with heavy metal-polluted filter dust and (iv) soil with both, heavy metal and acid rain treatment, over a period of 74 weeks. The mineralization of maize straw carbon was significantly reduced by heavy metal impact. Reduced mineralization rate of the added carbon likely resulted from a reduction of the microbial biomass due to heavy metal stress, while the efficiency of ¹³C incorporation into microbial PLFAs was hardly affected. Since acid rain did not significantly change soil pH, little impact on soil microorganisms and mineralization rate was found. Temporal dynamics of labelling of microbial PLFAs were different between bacterial and fungal PLFA biomarkers. Utilization of maize straw by bacterial PLFAs peaked immediately after the application (2 weeks), while labelling of the fungal biomarker 18:2ω6,9 was most pronounced 5 weeks after the application. In general, ¹³C labelling of microbial PLFAs was closely linked to the amounts of maize carbon present in the soil. The distinct higher labelling of microbial PLFAs in the heavy metal-polluted soils 74 weeks after application indicated a large fraction of available maize straw carbon still present in the soil.     

Variable use of plant- and soil-derived carbon by microorganisms in agricultural soils

In this study we used compound specific ¹³C and ¹⁴C isotopic signatures to determine the degree to which recent plant material and older soil organic matter (SOM) served as carbon substrates for microorganisms in soils. We determined the degree to which plant-derived carbon was used as a substrate by comparison of the ¹³C content of microbial phospholipid fatty acids (PLFA) from soils of two sites that had undergone a vegetation change from C3 to C4 plants in the past 20-30 years. The importance of much older SOM as a substrate was determined by comparison of the radiocarbon content of PLFA from soils of two sites that had different ¹⁴C concentrations of SOM.The ¹³C shift in PLFA from the two sites that had experienced different vegetation history indicated that 40-90% of the PLFA carbon had been fixed since the vegetation change took place. Thus PLFA were more enriched in ¹³C from the new C4 vegetation than it was observed for bulk SOM indicating recent plant material as preferentially used substrate for soil microorganisms. The largest ¹³C shift of PLFA was observed in the soil that had high ¹⁴C concentrations of bulk SOM. These results reinforce that organic carbon in this soil for the most part cycles rapidly. The degree to which SOM is incorporated into microbial PLFA was determined by the difference in ¹⁴C concentration of PLFA derived from two soils one with high ¹⁴C concentrations of bulk SOM and one with low. These results showed that 0-40% of SOM carbon is used as substrate for soil microorganisms. Furthermore a different substrate usage was identified for different microorganisms. Gram-negative bacteria were found to prefer recent plant material as microbial carbon source while Gram-positive bacteria use substantial amounts of SOM carbon. This was indicated by ¹³C as well as ¹⁴C signatures of their PLFA. Our results find evidence to support ‘priming’ in that PLFA indicative of Gram-negative bacteria associated with roots contain both plant- and SOM-derived C. Most interestingly, we find PLFA indicative of archeobacteria (methanothrophs) that may indicate the use of other carbon sources than plant material and SOM to a substantial amount suggesting that inert or slow carbon pools are not essential to explain carbon dynamics in soil.     

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.     

Comparison of soil fungal/bacterial ratios in a pH gradient using physiological and PLFA-based techniques

We have compared the total microbial biomass and the fungal/bacterial ratio estimated using substrate-induced respiration (SIR) in combination with the selective inhibition technique and using the phospholipid fatty acid (PLFA) technique in a pH gradient (3.0-7.2) consisting of 53 mature broad-leaved forest soils. A fungal/bacterial biomass index using the PLFA technique was calculated using the PLFA 18:2ω6,9 as an indicator of fungal biomass and the sum of 13 bacterial specific PLFAs as indicator of the bacterial biomass. Good linear correlation (p<0.001) was found between the total microbial biomass estimated with SIR and total PLFAs (totPLFA), indicating that 1 mg biomass-C was equivalent to 130 nmol totPLFA. Both biomass estimates were positively correlated to soil pH. The fungal/bacterial ratio measured using the selective inhibition technique decreased significantly with increasing pH from about 9 at pH 3 to approximately 2 at pH 7, while the fungal/bacterial biomass index using PLFA measurements tended to increase slightly with increasing soil pH. Good correlation between the soil content of ergosterol and of the PLFA 18:2ω6,9 indicated that the lack of congruency between the two methods in estimating fungal/bacterial ratios was not due to PLFA 18:2ω6,9-related non-fungal structures to any significant degree. Several PLFAs were strongly correlated to soil pH (R² values >0.8); for example the PLFAs 16:1ω5 and 16:1ω7c increased with increasing soil pH, while i16:0 and cy19:0 decreased. A principal component analysis of the total PLFA pattern gave a first component that was strongly correlated to soil pH (R²=0.85, p<0.001) indicating that the microbial community composition in these beech/beech-oak forest soils was to a large extent determined by soil pH.     

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.     

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.     

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.     

13C标记磷脂脂肪酸分析在土壤微生物生态 研究中的应用

磷脂脂肪酸(PLFA)是微生物细胞膜的重要组成成分,不同微生物群落可通过不同生化途径合成不同的PLFA,因此可选择某些PLFA作为微生物群落结构变化的生物标志物。PLFA与稳定性同位素~(13)C标记(~(13)C-PLFA)技术结合,不仅能够确定原位土壤环境中微生物群落组成,而且能够定向发掘土壤生态系统中参与碳源代谢过程的微生物群落,提供复杂群落中土壤微生物相互作用的信息,具有广阔的应用前景。其基本原理为:将富集~(13)C稳定同位素的基质加入土壤中,土壤中的某些微生物群落利用基质~(13)C合成PLFA,提取并纯化土壤微生物的PLFA,利用气相色谱-燃烧-同位素比例质谱(GC-C-IRMS)测定其~(13)C丰度,通过对比分析,从而获取微生物群落组成与其功能的直接信息。本文在介绍了~(13)C-PLFA原理的基础上,综述了该技术在光合同化碳的根际微生物利用、土壤有机质分解的激发效应、甲烷氧化、有机污染物降解、外源简单碳源和外源复杂碳源的微生物利用等方面的应用,对此项技术的优缺点进行了分析并展望了其未来应用。     

Residue chemistry and microbial community structure during decomposition of eucalypt, wheat and vetch residues

Previous studies have shown that residue chemistry and microbial community structure change during decomposition, however little is known about the relationship between C-chemistry and microbial community structure. To address this knowledge gap, we studied C-chemistry and microbial community structure during the decomposition of eucalypt, wheat and vetch residues with and without additional inorganic N. Bags containing ground residues of eucalypt, wheat, and vetch were buried in sand microcosms after inoculation with a diverse microbial community. Respiration was measured over an incubation period of 150 days. At different times during incubation, total C and N of the residues were analysed and residue carbon chemistry was determined by ¹³C-NMR (nuclear magnetic resonance) spectroscopy. Microbial communities were assessed by phospholipid fatty acid (PLFA) analyses.Results indicated that during decomposition, residue C-chemistry and microbial community composition changed over time and differed between residue types. Changes in microbial community structure were associated with changes in residue C-chemistry, mainly the relative content of aryl-C and O-alkyl-C. Addition of N increased cumulative respiration, altered C-chemistry during decomposition, particularly in high C/N residues (wheat and eucalypt), and changed microbial succession leading to an earlier establishment of a stable microbial community structure. N addition to eucalypt and wheat reduced the decomposition of aryl-C compounds.     

The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil

The cell content of 12 bacterial phospholipid fatty acids (PLFA) was determined in bacteria extracted from soil by homogenization/centrifugation. The bacteria were enumerated using acridine orange direct counts. An average of 1.40×10^-17 mol bacterial PLFA cell^-1 was found in bacteria extracted from 15 soils covering a wide range of pH and organic matter contents. With this factor, the bacterial biomass based on PLFA analyses of whole soil samples was calculated as 1.0–4.8 mg bacterial C g^-1 soil C. The corresponding range based on microscopical counts was 0.3–3.0 mg bacterial C g^-1 soil C. The recovery of bacteria from the soils using homogenization/centrifugation was 2.6–16% (mean 8.7%) measured by PLFA analysis, and 12–61% (mean 26%) measured as microscopical counts. The soil content of the PLFA 18:26 was correlated with the ergosterol content (r=0.92), which supports the use of this PLFA as an indicator of fungal biomass. The ratio 18:26 to bacterial PLFA is therefore suggested as an index of the fungal:bacterial biomass ratio in soil. An advantage with the method based on PLFA analyses is that the same technique and even the same sample is used to determine both fungi and bacteria. The fungal:bacterial biomass ratio calculated in this way was positively correlated with the organic matter content of the soils (r=0.94).     

Investigation of the effect of ammonium sulfate on populations of ambient methane oxidising bacteria by C-labelling and GC/C/IRMS analysis of phospholipid fatty acids

The oxidation of atmospheric methane by methanotrophic bacteria residing in soils constitutes an important terrestrial methane sink with previous studies having revealed the inhibition of microbially mediated methane oxidation in the presence of salt ions. The bacteria responsible for ambient methane oxidation are not amenable to currently available methods of culturing, resulting in the need for a method of in situ analysis. A combination of phospholipid fatty acid (PLFA) analysis and stable isotopic labelling has been employed in this investigation as a means of cultivation-independent bacterial analysis. Soil samples were treated with an ammonium sulfate solution at a concentration that was known to inhibit methane oxidation or with distilled water, serving as a control, and incubated with ¹³C-labelled methane. PLFAs were analysed by GC/C/IRMS in order to determine their ¹³C content and, hence, the PLFA distribution of the methane oxidising bacteria. Ammonium sulfate treatment reduced the amount of ¹³C incorporated into the majority of PLFAs except the i17:0 PLFA in the presence of high concentrations of methane. These results implied a shift in the composition of the methane oxidising bacterial community in the soils treated with ammonium ions, with the treatment appearing to suppress one group of organisms more than another.