Atmospheric CO2 enrichment and nutrient additions to planted soil increase mineralisation of soil organic matter, but do not alter microbial utilisation of plant- and soil C-sources

Plants link atmospheric and soil carbon pools through CO₂ fixation, carbon translocation, respiration and rhizodeposition. Within soil, microbial communities both mediate carbon-sequestration and return to the atmosphere through respiration. The balance of microbial use of plant-derived and soil organic matter (SOM) carbon sources and the influence of plant-derived inputs on microbial activity are key determinants of soil carbon-balance, but are difficult to quantify. In this study we applied continuous ¹³C-labelling to soil-grown Lolium perenne, imposing atmospheric CO₂ concentrations and nutrient additions as experimental treatments. The relative use of plant- and SOM-carbon by microbial communities was quantified by compound-specific ¹³C-analysis of phospholipid fatty acids (PLFAs). An isotopic mass-balance approach was applied to partition the substrate sources to soil respiration (i.e. plant- and SOM-derived), allowing direct quantification of SOM-mineralisation. Increased CO₂ concentration and nutrient amendment each increased plant growth and rhizodeposition, but did not greatly alter microbial substrate use in soil. However, the increased root growth and rhizosphere volume with elevated CO₂ and nutrient amendment resulted in increased rates of SOM-mineralisation per experimental unit. As rhizosphere microbial communities utilise both plant- and SOM C-sources, the results demonstrate that plant-induced priming of SOM-mineralisation can be driven by factors increasing plant growth. That the balance of microbial C-use was not affected on a specific basis may suggest that the treatments did not affect soil C-balance in this study.     

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.     

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.     

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.     

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.     

Three‐source partitioning of CO2 efflux from maize field soil by 13C natural abundance

A natural‐¹³C‐labeling approach—formerly observed under controlled conditions—was tested in the field to partition total soil CO₂ efflux into root respiration, rhizomicrobial respiration, and soil organic matter (SOM) decomposition. Different results were expected in the field due to different climate, site, and microbial properties in contrast to the laboratory. Within this isotopic method, maize was planted on soil with C₃‐vegetation history and the total CO₂ efflux from soil was subdivided by isotopic mass balance. The C₄‐derived C in soil microbial biomass was also determined. Additionally, in a root‐exclusion approach, root‐ and SOM‐derived CO₂ were determined by the total CO₂ effluxes from maize (Zea mays L.) and bare‐fallow plots. In both approaches, maize‐derived CO₂ contributed 22% to 35% to the total CO₂ efflux during the growth period, which was comparable to other field studies. In our laboratory study, this CO₂ fraction was tripled due to different climate, soil, and sampling conditions. In the natural‐¹³C‐labeling approach, rhizomicrobial respiration was low compared to other studies, which was related to a low amount of C₄‐derived microbial biomass. At the end of the growth period, however, 64% root respiration and 36% rhizomicrobial respiration in relation to total root‐derived CO₂ were calculated when considering high isotopic fractionations between SOM, microbial biomass, and CO₂. This relationship was closer to the 50% : 50% partitioning described in the literature than without fractionation (23% root respiration, 77% rhizomicrobial respiration). Fractionation processes of ¹³C must be taken into account when calculating CO₂ partitioning in soil. Both methods—natural ¹³C labeling and root exclusion—showed the same partitioning results when ¹³C isotopic fractionation during microbial respiration was considered and may therefore be used to separate plant‐ and SOM‐derived CO₂ sources.     

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.     

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.     

Carbon fluxes in soil food webs of increasing complexity revealed by C labelling and C natural abundance

Soil food webs are mainly based on three primary carbon (C) sources: root exudates, litter, and recalcitrant soil organic matter (SOM). These C sources vary in their availability and accessibility to soil organisms, which could lead to different pathways in soil food webs. The presence of three C isotopes (¹²C, ¹³C and ¹⁴C) offers an unique opportunity to investigate all three C sources simultaneously. In a microcosm experiment we studied the effect of food web complexity on the utilization of the three carbon sources. We choose an incomplete three factorial design with (i) living plants, (ii) litter and (iii) food web complexity. The most complex food web consisted of autochthonous microorganisms, nematodes, collembola, predatory mites, endogeic and anecic earthworms. We traced C from all three sources in soil, in CO₂ efflux and in individual organism groups by using maize grown on soil developed under C₃ vegetation and application of ¹⁴C labelled ryegrass shoots as a litter layer. The presence of living plants had a much greater effect on C pathways than food web complexity. Litter decomposition, measured as ¹⁴CO₂ efflux, was decreased in the presence of living plants from 71% to 33%. However, living plants increased the incorporation of litter C into microbial biomass and arrested carbon in the litter layer and in the upper soil layer. The only significant effect of food web complexity was on the litter C distribution in the soil layers. In treatments with fungivorous microarthropods (Collembola) the incorporation of litter carbon into mineral soil was reduced. Root exudates as C source were passed through rhizosphere microorganisms to the predator level (at least to the third trophic level). We conclude that living plants strongly affected C flows, directly by being a source of additional C, and indirectly by modifying the existing C flows within the food web including CO₂ efflux from the soil and litter decomposition.     

Rhizodeposition-induced decomposition increases N availability to wild and cultivated wheat genotypes under elevated CO2

Elevated CO₂ may increase nutrient availability in the rhizosphere by stimulating N release from recalcitrant soil organic matter (SOM) pools through enhanced rhizodeposition. We aimed to elucidate how CO₂-induced increases in rhizodeposition affect N release from recalcitrant SOM, and how wild versus cultivated genotypes of wheat mediated differential responses in soil N cycling under elevated CO₂. To quantify root-derived soil carbon (C) input and release of N from stable SOM pools, plants were grown for 1 month in microcosms, exposed to ¹³C labeling at ambient (392 μmol mol⁻¹) and elevated (792 μmol mol⁻¹) CO₂ concentrations, in soil containing ¹⁵N predominantly incorporated into recalcitrant SOM pools. Decomposition of stable soil C increased by 43%, root-derived soil C increased by 59%, and microbial-¹³C was enhanced by 50% under elevated compared to ambient CO₂. Concurrently, plant ¹⁵N uptake increased (+7%) under elevated CO₂ while ¹⁵N contents in the microbial biomass and mineral N pool decreased. Wild genotypes allocated more C to their roots, while cultivated genotypes allocated more C to their shoots under ambient and elevated CO₂. This led to increased stable C decomposition, but not to increased N acquisition for the wild genotypes. Data suggest that increased rhizodeposition under elevated CO₂ can stimulate mineralization of N from recalcitrant SOM pools and that contrasting C allocation patterns cannot fully explain plant mediated differential responses in soil N cycling to elevated CO₂.