Root and rhizomicrobial respiration: A review of approaches to estimate respiration by autotrophic and heterotrophic organisms in soil

Partitioning the root‐derived CO₂ efflux from soil (frequently termed rhizosphere respiration) into actual root respiration (RR, respiration by autotrophs) and rhizomicrobial respiration (RMR, respiration by heterotrophs) is crucial in determining the carbon (C) and energy balance of plants and soils. It is also essential in quantifying C sources for rhizosphere microorganisms and in estimation of the C contributing to turnover of soil organic matter (SOM), as well as in linking net ecosystem production (NEP) and net ecosystem exchange (NEE). Artificial‐environment studies such as hydroponics or sterile soils yield unrealistic C‐partitioning values and are unsuitable for predicting C flows under natural conditions. To date, several methods have been suggested to separate RR and RMR in nonsterile soils: 1) component integration, 2) substrate‐induced respiration, 3) respiration by excised roots, 4) comparison of root‐derived ¹⁴CO₂ with rhizomicrobial ¹⁴CO₂ after continuous labeling, 5) isotope dilution, 6) model‐rhizodeposition technique, 7) modeling of ¹⁴CO₂ efflux dynamics, 8) exudate elution, and 9) δ¹³C of CO₂ and microbial biomass. This review describes the basic principles and assumptions of these methods and compares the results obtained in the original papers and in studies designed to compare the methods. The component‐integration method leads to strong disturbance and non‐proportional increase of CO₂ efflux from different sources. Four of the methods (5 to 8) are based on the pulse labeling of shoots in a ¹⁴CO₂ atmosphere and subsequent monitoring of ¹⁴CO₂ efflux from the soil. The model‐rhizodeposition technique and exudate‐elution procedure strongly overestimate RR and underestimate RMR. Despite alternative assumptions, isotope dilution and modeling of ¹⁴CO₂‐efflux dynamics yield similar results. In crops and grasses (wheat, ryegrass, barley, buckwheat, maize, meadow fescue, prairie grasses), RR amounts on average to 48±5% and RMR to 52±5% of root‐derived CO₂. The method based on the ¹³C isotopic signature of CO₂ and microbial biomass is the most promising approach, especially when the plants are continuously labeled in ¹³CO₂ or ¹⁴CO₂ atmosphere. The “difference” methods, i.e., trenching, tree girdling, root‐exclusion techniques, etc., are not suitable for separating the respiration by autotrophic and heterotrophic organisms because the difference methods neglect the importance of microbial respiration of rhizodeposits.     

Microbial immobilisation of C rhizodeposits in rhizosphere and root-free soil under continuous C labelling of oats

A greenhouse experiment was conducted by growing oats (Avenasativa L.) in a continuously ¹³CO₂ labeled atmosphere. The allocation of ¹³C-labeled photosynthates in plants, microbial biomass in rhizosphere and root-free soil, pools of soil organic C, and CO₂ emissions were examined over the plant's life cycle. To isolate rhizosphere from root-free soil, plant seedlings were placed into bags made of nylon monofilament screen tissue (16 μm mesh) filled with soil. Two peaks of ¹³C in rhizosphere pools of microbial biomass and dissolved organic carbon (DOC), as well as in CO₂ emissions at the earing and ripeness stages were revealed. These ¹³C maxima corresponded to: (i) the end of rapid root growth and (ii) beginning of root decomposition, respectively. The δ¹³C values of microbial biomass were higher than those of DOC and of soil organic matter (SOM). The microbial biomass C accounted for up to 56 and 39% of ¹³C recovered in the rhizosphere and root-free soil, respectively. Between 4 and 28% of ¹³C assimilated was recovered in the root-free soil. Depending on the phenological stage, the contribution of root-derived C to total CO₂ emission from soil varied from 61 to 92% of total CO₂ evolved, including 4-23% attributed to rhizomicrobial respiration. While 81-91% of C substrates used for microbial growth in the root-free soil and rhizosphere came from SOM, the remaining 9-19% of C substrates utilized by the microbial biomass was attributable to rhizodeposition. The use of continuous isotopic labelling and physical separation of root-free and rhizosphere soil, combined with natural ¹³C abundance were effective in gaining new insight on soil and rhizosphere C-cycling.     

Three-source partitioning of CO2 efflux from soil planted with maize by C natural abundance fails due to inactive microbial biomass

A theoretical approach to the partitioning of carbon dioxide (CO₂) efflux from soil with a C₃ vegetation history planted with maize (Zea mays), a C₄ plant, into three sources, root respiration (RR), rhizomicrobial respiration (RMR), and microbial soil organic matter (SOM) decomposition (SOMD), was examined. The δ¹³C values of SOM, roots, microbial biomass, and total CO₂ efflux were measured during a 40-day growing period. A three-source isotopic mass balance based on the measured δ¹³C values and on assumptions made in other studies showed that RR, RMR, and SOMD amounted to 91%, 4%, and 5%, respectively. Two assumptions were thoroughly examined in a sensitivity analysis: the absence of ¹³C fractionation and the conformity of δ¹³C of microbial CO₂ and that of microbial biomass. This approach strongly overestimated RR and underestimated RMR and microbial SOMD. CO₂ efflux from unplanted soil was enriched in ¹³C by 2.0‰ compared to microbial biomass. The consideration of this ¹³C fractionation in the mass balance equation changed the proportions of RR and RMR by only 4% and did not affect SOMD. A calculated δ¹³C value of microbial CO₂ by a mass balance equation including active and inactive parts of microbial biomass was used to adjust a hypothetical below-ground CO₂ partitioning to the measured and literature data. The active microbial biomass in the rhizosphere amounted to 37% to achieve an appropriate ratio between RR and RMR compared to measured data. Therefore, the three-source partitioning approach failed due to a low active portion of microbial biomass, which is the main microbial CO₂ source controlling the δ¹³C value of total microbial biomass. Since fumigation-extraction reflects total microbial biomass, its δ¹³C value was unsuitable to predict δ¹³C of released microbial CO₂ after a C₃-C₄ vegetation change. The second adjustment to the CO₂ partitioning results in the literature showed that at least 71% of the active microbial biomass utilizing maize rhizodeposits would be necessary to achieve that proportion between RR and RMR observed by other approaches based on ¹⁴C labelling. The method for partitioning total below-ground CO₂ efflux into three sources using a natural ¹³C labelling technique failed due to the small proportion of active microbial biomass in the rhizosphere. This small active fraction led to a discrepancy between δ¹³C values of microbial biomass and of microbially respired CO₂.     

Sources of CO2 efflux from soil and review of partitioning methods

Five main biogenic sources of CO₂ efflux from soils have been distinguished and described according to their turnover rates and the mean residence time of carbon. They are root respiration, rhizomicrobial respiration, decomposition of plant residues, the priming effect induced by root exudation or by addition of plant residues, and basal respiration by microbial decomposition of soil organic matter (SOM). These sources can be grouped in several combinations to summarize CO₂ efflux from the soil including: root-derived CO₂, plant-derived CO₂, SOM-derived CO₂, rhizosphere respiration, heterotrophic microbial respiration (respiration by heterotrophs), and respiration by autotrophs. These distinctions are important because without separation of SOM-derived CO₂ from plant-derived CO₂, measurements of total soil respiration have very limited value for evaluation of the soil as a source or sink of atmospheric CO₂ and for interpreting the sources of CO₂ and the fate of carbon within soils and ecosystems. Additionally, the processes linked to the five sources of CO₂ efflux from soil have various responses to environmental variables and consequently to global warming. This review describes the basic principles and assumptions of the following methods which allow SOM-derived and root-derived CO₂ efflux to be separated under laboratory and field conditions: root exclusion techniques, shading and clipping, tree girdling, regression, component integration, excised roots and insitu root respiration; continuous and pulse labeling, ¹³C natural abundance and FACE, and radiocarbon dating and bomb-¹⁴C. A short sections cover the separation of the respiration of autotrophs and that of heterotrophs, i.e. the separation of actual root respiration from microbial respiration, as well as methods allowing the amount of CO₂ evolved by decomposition of plant residues and by priming effects to be estimated. All these methods have been evaluated according to their inherent disturbance of the ecosystem and C fluxes, and their versatility under various conditions. The shortfalls of existing approaches and the need for further development and standardization of methods are highlighted.     

Carbon input by plants into the soil. Review

The methods used for estimating below‐ground carbon (C) translocation by plants, and the results obtained for different plant species are reviewed. Three tracer techniques using C isotopes to quantify root‐derived C are discussed: pulse labeling, continuous labeling, and a method based on the difference in ¹³C natural abundance in C3 and C4 plants. It is shown, that only the tracer methods provided adequate results for the whole below‐ground C translocation. This included roots, exudates and other organic substances, quickly decomposable by soil microorganisms, and CO₂ produced by root respiration. Advantages due to coupling of two different tracer techniques are shown. The differences in the below‐ground C translocation pattern between plant species (cereals, grasses, and trees) are discussed. Cereals (wheat and barley) transfer 20%—30% of total assimilated C into the soil. Half of this amount is subsequently found in the roots and about one‐third in CO₂ evolved from the soil by root respiration and microbial utilization of rootborne organic substances. The remaining part of below‐ground translocated C is incorporated into the soil microorganisms and soil organic matter. The portion of assimilated C allocated below the ground by cereals decreases during growth and by increasing N fertilization. Pasture plants translocated about 30%—50% of assimilates below‐ground, and their translocation patterns were similar to those of crop plants. On average, the total C amounts translocated into the soil by cereals and pasture plants are approximately the same (1500 kg C ha^—1), when the same growth period is considered. However, during one vegetation period the cereals and grasses allocated beneath the ground about 1500 and 2200 kg C ha^—1, respectively. Finally, a simple approach is suggested for a rough calculation of C input into the soil and for root‐derived CO₂ efflux from the soil.     

Carbon turnover in the rhizosphere under continuous plant labeling with <Superscript>13</Superscript>CO<Subscript>2</Subscript>: Partitioning of root,microbial, and rhizomicrobial respiration

The input of labeled C into the pool of soil organic matter, the CO₂ fluxes from the soil, and the contribution of root and microbial respiration to the CO₂ emission were studied in a greenhouse experiment with continuous labeling of oat plants with ¹³CO₂ using the method of the natural ¹³C abundance in the air. The carbon of the microbial biomass composed 56 and 39% of the total amounts of ¹³C photoassimilates in the rhizosphere and in the bulk soil, respectively. The contribution of root respiration to the CO₂ emission from the soil reached 61–92%, including 4–23% of the rhizomicrobial respiration. The contribution of the microbial respiration to the total CO₂ emission from the soil varied from 8 to 39%. The soil organic matter served as the major carbon-containing substrate for microorganisms in the bulk soil and in the rhizosphere: 81–91% of the total amount of carbon involved in the microbial metabolism was derived from the soil organic matter.     

Partitioning CO2 Respiration among Soil,Rhizosphere Microorganisms,and Roots of Wheat under Greenhouse Conditions

Abstract

The measurement of soil, root, and rhizomicrobial respiration has become very important in evaluating the role of soil on atmospheric carbon dioxide (CO₂) concentration. The objective of this study was to partition root, rhizosphere, and nonrhizosphere soil respiration during wheat growth. A secondary objective was to compare three techniques for measuring root respiration: without removing shoot of wheat, shading shoot of wheat, and removing shoot of wheat. Soil, root, and rhizomicrobial respiration were determined during wheat growth under greenhouse conditions in a Carwile loam soil (fine, mixed, superactive, thermic Typic Argiaquolls). Total below ground respiration from planted pots increased after planting through early boot stage and then decreased through physiological maturity. Root‐rhizomicrobial respiration was determined by taking the difference in CO₂ flux between planted and unplanted pots. Also, root and rhizomicrobial respirations were directly measured from roots by placing them inside a Mason jar. It was determined that root‐rhizomicrobial respiration accounted for 60% of total CO₂ flux, whereas 40% was from heterotrophic respiration in unplanted pots. Rhizomicrobial respiration accounted for 18 to 25% of total CO₂ flux. Shade and no‐shoot had similar effects on root respiration. The three techniques were not significantly different (p>0.05).     

Isotopic analysis of respired CO2 during decomposition of separated soil organic matter pools

A detailed understanding of the processes that contribute to the δ¹³C value of respired CO₂ is necessary to make links between the isotopic signature of CO₂ efflux from the soil surface and various sources within the soil profile. We used density fractionation to divide soils from two forested sites that are a part of an ongoing detrital manipulation experiment (the Detrital Input and Removal Treatments, or DIRT project) into two soil organic matter pools, each of which contributes differently to total soil CO₂ efflux. In both sites, distinct biological pools resulted from density fractionation; however, our results do not always support the concept that the light fraction is readily decomposable whereas the heavy fraction is recalcitrant. In a laboratory incubation following density fractionation we found that cumulative respiration over the course of the incubation period was greater from the light fraction than from the heavy fraction for the deciduous site, while the opposite was true for the coniferous site.Use of stable isotopes yielded insight as to the nature of the density fractions, with the heavy fraction solids from both forests isotopically enriched relative to those of the light fraction. The isotopic signature of respired CO₂, however, was more complicated. During incubation of the fractions there was an initial isotopic depletion of the respired CO₂ compared to the substrate for both soil fractions from both forests. Over time for both fractions of both soils the respired δ¹³C reflected more closely the initial substrate value; however, the transition from depleted to enriched respiration relative to substrate occurs at a different stage of decomposition depending on site and substrate recalcitrance. We found a relationship between cumulative respiration during the incubation period and the duration of the transition from isotopically depleted to enriched respiration in the coniferous site but not the deciduous site. Our results suggest that a shift in microbial community or to dead microbial biomass as a substrate could be responsible for the transition in the isotopic signature of respired CO₂ during decomposition. It is likely that a combination of organic matter quality and isotopic discrimination by microbes, in addition to differences in microbial community composition, contribute to the isotopic signature of different organic matter fractions. It is apparent that respired δ¹³CO₂ cannot be assumed to be a direct representation of the substrate δ¹³C. Detailed knowledge of the soil characteristics at a particular site is necessary to interpret relationships between the isotopic values of a substrate and respired CO₂.     

Carbon flows in the rhizosphere of ryegrass (Lolium perenne)

This study addresses the issue of carbon (C) fluxes through below ground pools within the rhizosphere of Lolium perenne using the ¹⁴C pulse labeling. Lolium perenne was grown in plexiglas chambers on topsoil of a Haplic Luvisol under controled laboratory conditions. ¹⁴C‐CO₂ efflux from soil, as well as ¹⁴C content in shoots, roots, soil, dissolved organic C (DOC), and microbial biomass were monitored for 11 days after the pulsing. Lolium allocates about 48 % of the total assimilated ¹⁴C below the soil surface, and roots were the primary sink for this C. Maximum ¹⁴C content in the roots was observed 12 hours after the labeling and it amounts to 42 % of the assimilated C. Only half of the ¹⁴C amount was found in the roots at the end of the monitoring period. The remainder was lost through root respiration, root decomposition, and rhizodeposition. Six hours after the ¹⁴C pulse labeling soil accounted for 11 %, DOC for 1.1 %, and microbial biomass for 4.9 % of assimilated C. ¹⁴C in CO₂ efflux from soil was detected as early as 30 minutes after labeling. The maximum ¹⁴C‐CO₂ emission rate (0.34 % of assimilated ¹⁴C h^—1) from the soil occurred between four and twelve hours after labeling. From the 5^th day onwards, only insignificant changes in carbon partitioning occurred. The partitioning of assimilated C was completed after 5 days after assimilation. Based on the ¹⁴C partitioning pattern, we calculated the amount of assimilated C during 47 days of growth at 256 g C m^—2. Of this amount 122 g C m^—2 were allocated to below ground, shoots retained 64 g C m^—2, and 70 g C m^—2 were lost from the shoots due to respiration. Roots were the main sink for below ground C and they accounted for 74 g C m^—2, while 28 g C m^—2 were respired and 19 g C m^—2 were found as residual ¹⁴C in soil and microorganisms.     

Contribution of rhizomicrobial and root respiration to the CO2 emission from soil (A review)

Separate determination of root respiration and rhizomicrobial respiration is one of the most interesting, important, and methodologically complicated problems in the study of the carbon budget in soils and the subdivision of the CO₂ emission from soils into separate fluxes. In this review, we compare the main principles, the advantages and disadvantages, and the results obtained by the methods of component integration, substrate-induced respiration, respiratory capacity, girdling, isotope dilution, model rhizodeposition, modeling of the ¹⁴CO₂ efflux dynamics, exudates elution, and the δ¹³C measurements of the microbial biomass and CO₂. Summarizing the results of the determinations performed by these methods, we argue that about 40% of the rhizosphere CO₂ efflux is due to root respiration and about 60% of this efflux is due to the respiration of microorganisms decomposing root exudates.     

Active microbial RNA turnover in a grassland soil estimated using a CO2 spike

Rhizosphere microbes are critical to the initial transfer and transformation of root carbon inputs to the soil but our understanding of the activity of these organisms remains constrained by their limited culturability. In this study we combined isotopic ¹³C tracer and molecular approaches to measure the incorporation of recently assimilated plant C into soil microbial RNA and DNA pools as a means to determine the turnover of the ‘active’ rhizosphere community. This required the development of a method for the extraction, purification and preparation of small-sample soil DNA and RNA (<5 μg C) for isotope analysis. Soil, plant and respired CO₂ samples were collected from a ¹³CO₂ pulse-chase experiment at intervals for 20 days post-labelling. The peak of ¹³C release in soil/root respired CO₂ came between 5 and 48 h after ¹³CO₂ pulse-labelling and was followed by a secondary peak of soil heterotroph ¹³C respiration after 136 h. Results showed that both soil DNA and RNA rapidly incorporated recent photosynthate with greatest ¹³C found in the ‘active’ microbial RNA fraction reflecting higher rates of microbial RNA turnover. The dilution rate of the pulse derived ¹³C in RNA-C was used to estimate a microbial RNA turnover of approximately 20% day⁻¹ with a 15-20 day residence time for photosynthate derived ¹³C in the RNA pool. The findings of this work confirm the rapid transfer of photosynthate C inputs through soil microorganisms to the atmosphere as CO₂ and the potential of the biomolecular-isotope tracer approach in soil C research.     

The Origin of Soil Organic C,Dissolved Organic C and Respiration in a Long‐Term Maize Experiment in Halle,Germany, Determined by 13C Natural Abundance

For a quantitative analysis of SOC dynamics it is necessary to trace the origins of the soil organic compounds and the pathways of their transformations. We used the ¹³C isotope to determine the incorporation of maize residues into the soil organic carbon (SOC), to trace the origin of the dissolved organic carbon (DOC), and to quantify the fraction of the maize C in the soil respiration. The maize‐derived SOC was quantified in soil samples collected to a depth of 65 cm from two plots, one ’︁continuous maize’ and the other ’︁continuous rye’ (reference site) from the long‐term field experiment ’︁Ewiger Roggen’ in Halle. This field trial was established in 1878 and was partly changed to a continuous maize cropping system in 1961. Production rates and δ¹³C of DOC and CO₂ were determined for the Ap horizon in incubation experiments with undisturbed soil columns. After 37 years of continuous maize cropping, 15% of the total SOC in the topsoil originated from maize C. The fraction of the maize‐derived C below the ploughed horizon was only 5 to 3%. The total amount of maize C stored in the profile was 9080 kg ha⁻¹ which was equal to about 31% of the estimated total C input via maize residues (roots and stubble). Total leaching of DOC during the incubation period of 16 weeks was 1.1 g m⁻² and one third of the DOC derived from maize C. The specific DOC production rate from the maize‐derived SOC was 2.5 times higher than that from the older humus formed by C₃ plants. The total CO₂‐C emission for 16 weeks was 18 g m⁻². Fifty‐eight percent of the soil respiration originated from maize C. The specific CO₂ formation from maize‐derived SOC was 8 times higher than that from the older SOC formed by C₃ plants. The ratio of DOC production to CO₂‐C production was three times smaller for the young, maize‐derived SOC than for the older humus formed by C₃ plants.     

Impact of plant species and atmospheric CO2 concentration on rhizodeposition and soil microbial activity and community composition

Both plant species and CO₂ concentration can potentially affect rhizodeposition and consequently soil microbial activity and community composition. However, the effect differs based on plant developmental stage. We focused on the effect of three plant species (forbs, grasses, and N₂‐fixers) at an early stage of development on root C deposition and fate, soil organic matter (SOM) mineralization and soil microbial community composition at ambient (aCO₂) and elevated (eCO₂) CO₂ levels. Plants were grown from seed, under continuous ¹³C‐labelling atmospheres (400 and 800 µmol mol^?1 CO₂), in grassland soil for three weeks. At the end of the growth period, soil respiration, dissolved organic C (DOC) and phospholipid fatty acid (PLFA) profiles were quantified and isotopically partitioned into root‐ and soil‐derived components. Root‐derived DOC (0.53 ± 0.34 and 0.26 ± 0.29 µg mL soil solution^?1) and soil‐derived CO₂ (6.14 ± 0.55 and 5.04 ± 0.44 µg CO₂‐C h^?1) were on average two times and 22% higher at eCO₂ than at aCO₂, respectively. Plant species differed in exudate production at aCO₂ (0.11 ± 0.11, 0.10 ± 0.18, and 0.58 ± 0.58 µg mL soil solution^?1 for Plantago, Festuca, and Lotus, respectively) but not at eCO₂ (0.20 ± 0.28, 0.66 ± 0.32, and 0.75 ± 0.15 µg mL soil solution^?1 for Plantago, Festuca, and Lotus, respectively). However, no differences among plant species or CO₂ levels were apparent when DOC was expressed per gram of roots. Relative abundance of PLFAs did not differ between the two CO₂ levels. A higher abundance of actinobacteria and G‐positive bacteria occurred in unplanted (8.07 ± 0.48 and 24.36 ± 1.18 mol%) and Festuca‐affected (7.63 ± 0.31 and 23.62 ± 0.69 mol%) soil than in Plantago‐ (7.04 ± 0.36 and 23.41 ± 1.13 mol%) and Lotus‐affected (7.24 ± 0.17 and 23.13 ± 0.52 mol%) soil. In conclusion, the differences in root exudate production and soil respiration are mainly caused by differences in root biomass at an early stage of development. However, plant species evidently produce root exudates of varying quality affecting associated microbial community composition.     

Plant biomass influences rhizosphere priming effects on soil organic matter decomposition in two differently managed soils

We used a continuous labeling method of naturally ¹³C-depleted CO₂ in a growth chamber to test for rhizosphere effects on soil organic matter (SOM) decomposition. Two C3 plant species, soybean (Glycine max) and sunflower (Helianthus annus), were grown in two previously differently managed soils, an organically farmed soil and a soil from an annual grassland. We maintained a constant atmospheric CO₂ concentration at 400±5 ppm and δ¹³C signature at −24.4‰ by regulating the flow of naturally ¹³C-depleted CO₂ and CO₂-free air into the growth chamber, which allowed us to separate new plant-derived CO₂-C from original soil-derived CO₂-C in soil respiration. Rhizosphere priming effects on SOM decomposition, i.e., differences in soil-derived CO₂-C between planted and non-planted treatments, were significantly different between the two soils, but not between the two plant species. Soil-derived CO₂-C efflux in the organically farmed soil increased up to 61% compared to the no-plant control, while the annual grassland soil showed a negligible increase (up to 5% increase), despite an overall larger efflux of soil-derived CO₂-C and total soil C content. Differences in rhizosphere priming effects on SOM decomposition between the two soils could be largely explained by differences in plant biomass, and in particular leaf biomass, explaining 49% and 74% of the variation in primed soil C among soils and plant species, respectively. Nitrogen uptake rates by soybean and sunflower was relatively high compared to soil C respiration and associated N mineralization, while inorganic N pools were significantly depleted in the organic farm soil by the end of the experiment. Despite relatively large increases in SOM decomposition caused by rhizosphere effects in the organic farm soil, the fast-growing soybean and sunflower plants gained little extra N from the increase in SOM decomposition caused by rhizosphere effects. We conclude that rhizosphere priming effects of annual plants on SOM decomposition are largely driven by plant biomass, especially in soils of high fertility that can sustain high plant productivity.     

C fractionation at the root-microorganisms-soil interface: A review and outlook for partitioning studies

Natural variations of the ¹³C/¹²C ratio have been frequently used over the last three decades to trace C sources and fluxes between plants, microorganisms, and soil. Many of these studies have used the natural-¹³C-labelling approach, i.e. natural δ¹³C variation after C₃-C₄ vegetation changes. In this review, we focus on ¹³C fractionation in main processes at the interface between roots, microorganisms, and soil: root respiration, microbial respiration, formation of dissolved organic carbon, as well as microbial uptake and utilization of soil organic matter (SOM). Based on literature data and our own studies, we estimated that, on average, the roots of C₃ and C₄ plants are ¹³C enriched compared to shoots by +1.2 ± 0.6‰ and +0.3 ± 0.4‰, respectively. The CO₂ released by root respiration was ¹³C depleted by about −2.1 ± 2.2‰ for C₃ plants and −1.3 ± 2.4‰ for C₄ plants compared to root tissue. However, only a very few studies investigated ¹³C fractionation by root respiration. This urgently calls for further research. In soils developed under C₃ vegetation, the microbial biomass was ¹³C enriched by +1.2 ± 2.6‰ and microbial CO₂ was also ¹³C enriched by +0.7 ± 2.8‰ compared to SOM. This discrimination pattern suggests preferential utilization of ¹³C-enriched substances by microorganisms, but a respiration of lighter compounds from this fraction. The δ¹³C signature of the microbial pool is composed of metabolically active and dormant microorganisms; the respired CO₂, however, derives mainly from active organisms. This discrepancy and the preferential substrate utilization explain the δ¹³C differences between microorganisms and CO₂ by an ‘apparent’ ¹³C discrimination. Preferential consumption of easily decomposable substrates and less negative δ¹³C values were common for substances with low C/N ratios. Preferential substrate utilization was more important for C₃ soils because, in C₄ soils, microbial respiration strictly followed kinetics, i.e. microorganisms incorporated heavier C (? = +1.1‰) and respired lighter C (? = −1.1‰) than SOM. Temperature and precipitation had no significant effect on the ¹³C fractionation in these processes in C₃ soils. Increasing temperature and decreasing precipitation led, however, to increasing δ¹³C of soil C pools.Based on these ¹³C fractionations we developed a number of consequences for C partitioning studies using ¹³C natural abundance. In the framework of standard isotope mixing models, we calculated CO₂ partitioning using the natural-¹³C-labelling approach at a vegetation change from C₃ to C₄ plants assuming a root-derived fraction between 0% and 100% to total soil CO₂. Disregarding any ¹³C fractionation processes, the calculated results deviated by up to 10% from the assumed fractions. Accounting for ¹³C fractionation in the standard deviations of the C₄ source and the mixing pool did not improve the exactness of the partitioning results; rather, it doubled the standard errors of the CO₂ pools. Including ¹³C fractionations directly into the mass balance equations reproduced the assumed CO₂ partitioning exactly. At the end, we therefore give recommendations on how to consider ¹³C fractionations in research on carbon flows between plants, microorganisms, and soil.     

Root-derived respiration and non-structural carbon of rice seedlings

Various methods have been suggested to separate root and microbial contributions to soil respiration. However, to date there is no ideal approach available to partition below-ground CO₂ fluxes in its components although the combination of traditional methods with approaches based on isotopes seems especially promising for the future improvement of estimates. Here we provide evidence for the applicability of a new approach based on the hypothesis that root-derived (rhizomicrobial) respiration, including root respiration and CO₂ derived from microbial activity in the immediate vicinity of the root, is proportional to non-structural carbon contents (sugars and organic acids) of plant tissues. We examined relationships between root-derived CO₂ and non-structural carbon of rice (Oryza sativa) seedlings using ¹⁴C pulse labelling techniques, which partitioned the ¹⁴C fixed by photosynthesis into root-derived ¹⁴CO₂, and ¹⁴C in sugars and organic acids of roots and shoots. After the ¹⁴C pulse ¹⁴C in both sugars and organic acids of plant tissues decreased steeply during the first 12 h, and then decreased at a lower rate during the remaining 60 h. Soil ¹⁴CO₂ efflux and soil CO₂ efflux strongly depended on ¹⁴C pools in non-structural carbon of the plant tissues. Based on the linear regression between root-derived respiration and total non-structural carbon (sugars and organic acids) of roots, non-rhizomicrobial respiration (SOM-derived) was estimated to be 0.25 mg C g⁻¹ root d.w. h⁻¹. Assuming the value was constant, root-derived respiration contributed 85–92% to bulk soil respiration.     

Moisture modulates rhizosphere effects on C decomposition in two different soil types

While it is well known that soil moisture directly affects microbial activity and soil organic matter (SOM) decomposition, it is unclear if the presence of plants alters these effects through rhizosphere processes. We studied soil moisture effects on SOM decomposition with and without sunflower and soybean. Plants were grown in two different soil types with soil moisture contents of 45% and 85% of field capacity in a greenhouse experiment. We continuously labeled plants with depleted ¹³C, which allowed us to separate plant-derived CO₂-C from original soil-derived CO₂-C in soil respiration measurements. We observed an overall increase in soil-derived CO₂-C efflux in the presence of plants (priming effect) in both soils. On average a greater priming effect was found in the high soil moisture treatment (up to 76% increase in soil-derived CO₂-C compared to control) than in the low soil moisture treatment (up to 52% increase). Greater plant-derived CO₂-C and plant biomass in the high soil moisture treatment contributed to greater priming effects, but priming effects remained significantly higher in the high moisture treatment than in the low moisture treatment after correcting for the effects of plant-derived CO₂-C and plant biomass. The response to soil moisture particularly occurred in the sandy loam soil by the end of the experiment. Possibly, production of root exudates increased with increased soil moisture content. Root exudation of labile C may also have become more effective in stimulating microbial decomposition in the higher soil moisture treatment and sandy loam soil. Our results indicate that moisture conditions significantly modulate rhizosphere effects on SOM decomposition.     

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.     

Feldmethode zur Bestimmung der substrat‐induzierten Bodenatmung

A field method for the measurement of substrate‐induced soil respiration A novel method for in situ measurements of microbial soil activity using the CO₂ efflux combined with kinetic analysis is proposed. The results are compared with two conventional, laboratory methods, (1) substrate‐induced respiration using a ’︁Sapromat’ and (2) dehydrogenase activity. Soil respiration was measured in situ after addition of aqueous solutions containing 0 to 6 g glucose kg^—1 soil. The respiration data were analysed using kinetic models to describe the nutritional status of the soil bacteria employing few representative parameters. The two‐phase soil respiration response gave best fit results with the Hanes' or non‐parametric kinetic model with Michaelis‐Menten constants (K_m) of 0.05—0.1 g glucose kg^—1 soil. The maximum respiration rates (V_max) were obtained above 1 g glucose. Substrate‐induced respiration rates of the novel in situ method were significantly correlated to results of the ’︁Sapromat’ measurements (r² = 0.81***). The in situ method combined with kinetic analysis was suitable for the characterisation of microbial activity in soil; it showed respiration rates lower by 59% than measured in the laboratory with disturbed samples.     

Using natural 13C abundances to differentiate between three CO2 sources during incubation of a grassland soil amended with slurry and sugar

This study describes a novel approach to separate three soil carbon (C) sources by one tracer method (here ¹³C natural abundance). The approach uses the temporal dynamics of the CO₂ efflux from a C₃ grassland soil amended with added C₃ or C₄ slurry and/or C₃ or C₄ sugar to estimate contributions of three separate C sources (native soil, slurry, and sugar) to CO₂ efflux. Soil with slurry and/or sugar was incubated under controlled conditions, and concentration and δ¹³C values of evolved CO₂ were measured over a 2‐week period. The main assumption needed for separation of three C sources in CO₂ efflux, i.e., identical decomposition of applied C₃ and C₄ sugars in soil, was investigated and proven. The relative contribution to the CO₂ efflux was higher, but shorter with an increased (microbial) availability of the C source, i.e., sugar > slurry > SOM. The shortcomings and limitations as well as possible future applications of the suggested method are discussed.