Enzymes of Carbohydrate Metabolism in Leishmania donovani Amastigotes1

A method for the isolation of Leishmania donovani amastigotes from infected hamster spleen and liver tissues is described. Over 85% of the isolated amastigotes were viable as judged by acridine orange-ethidium bromide staining and in vitro transformation to the promastigote form. A comprehensive survey of the enzymes of carbohydrate metabolism in L. donovani amastigotes and promastigotes was conducted. Amastigotes and promastigotes possess all of the enzymes of the Embden-Meyerhof pathway, hexose monophosphate shunt, and tricarboxylic acid cycle. Cell-free extracts of both forms show pyruvate dehydrogenase activity which permits entry of pyruvate into the tricarboxylic acid cycle. Both forms demonstrate an active glutamate dehydrogenase, thus linking amino acid metabolism with carbohydrate metabolism. Pyruvate carboxylase, the enzyme responsible for replenishment of C₄ acids by heterotrophic CO₂ fixation into pyruvate, was also demonstrable in the tissue and insect forms. In general, activities of promastigote enzymes are higher than the amastigote enzymes. Differences between the vertebrate (amastigote) and invertebrate (promastigote) forms in their potential to utilize carbohydrates as substrates would appear to be quantitative rather than qualitative.     

Sequestration and turnover of bacterial- and fungal-derived carbon in a temperate grassland soil under long-term elevated atmospheric pCO2

Temperate grasslands contribute about 20% to the global C budget. Elevation of atmospheric CO₂ concentration (pCO₂) could lead to additional C sequestration into these ecosystems. Microbial‐derived C in the soil comprising about 1–5% of total soil organic carbon may be an important ‘pool’ for long‐term storage of C under future increased atmospheric CO₂ concentrations. In our study, the impact of elevated pCO₂ on bacterial‐ and fungal‐derived C in the soil of Lolium perenne pastures was investigated under free air carbon dioxide enrichment (FACE) conditions. For 7 years, L. perenne swards were exposed to ambient and elevated pCO₂ (36 and 60 Pa pCO₂, respectively). The additional CO₂ in the FACE plots was depleted in ¹³C compared with ambient plots, so that ‘new’ (<7 years) C inputs in the form of microbial‐derived residues could be determined by means of stable C isotope analysis. Amino sugars in soil are reliable organic biomarkers for indicating the presence of microbial‐derived residues, with particular amino sugars indicative of either bacterial or fungal origin. It is assumed that amino sugars are stabilized to a significant extent in soil, and so may play an important role in long‐term C storage. In our study, we were also able to discriminate between ‘old’ (> 7 years) and ‘new’ microbial‐derived C using compound‐specific δ¹³C analysis of individual amino sugars. This new tool was very useful in investigating the potential for C storage in microbial‐derived residues and the turnover of this C in soil under increased atmospheric pCO₂. The ¹³C signature of individual amino sugars varied between ?17.4‰ and ?39.6‰, and was up to 11.5% depleted in ¹³C in the FACE plots when compared with the bulk δ¹³C value of the native C3 L. perenne soil. New amino sugars in the bulk soil contributed up to 16% to the overall amino sugar pool after the first year and between 62% and 125% after 7 years of exposure to elevated pCO₂. Amounts of new glucosamine increased by the greatest amount (16–125%) during the experiment, followed by mannosamine (?9% to 107%), muramic acid (?11% to 97%), and galactosamine (15–62%). Proportions of new amino sugars in particle size fractions varied between 38% for muramic acid in the clay fraction and 100% for glucosamine and galactosamine in the coarse sand fraction. Summarizing, during the 7‐year period, amino sugars constituted only between 0.9% and 1.6% of the total SOC content. Therefore, their absolute significance for long‐term C sequestration is limited. Additionally new amino sugars were only sequestered in the silt fraction upon elevated pCO₂ exposure while amino sugar concentrations in the clay fraction decreased. Overall, amino sugar concentrations in bulk soil did not change significantly upon exposure to elevated pCO₂. The calculated mean residence time of amino sugars was surprisingly low varying between 6 and 90 years in the bulk soil, and between 3 and 30 years in the particle size fractions, representing soil organic matter pools with different but relatively low turnover times. Therefore, compound‐specific δ¹³C analysis of individual amino sugars clearly revealed a high amino sugar turnover despite more or less constant amino sugar concentrations over a 7 years period of exposure to elevated pCO₂.     

Mathematical modeling of soil carbon turnover in natural Podocarpus forest and Eucalyptus plantation in Ethiopia using compound specific δ13C analysis

Forest soils exhibit huge potential in storing carbon, but may also release large amounts of it if they undergo major changes in land use and environmental conditions. Biogeochemical processes controlling accumulation and release of soil organic carbon (SOC) are not yet sufficiently understood. We investigate the dynamics of SOC depending on its chemical composition below a natural forest (Podocarpus falcatus dominated) and a plantation (Eucalyptus saligna) growing on Nitisols in southern Ethiopia. Soils at the study‐site show a huge shift to less negative δ¹³C values at a depth of 20–30 cm, indicating a change from C4 savanna to C3 forest during the late Holocene. Total organic carbon (TOC), black carbon (BC), and sugars from microbial (rhamnose, fucose) and plant origin (xylose, arabinose) are subjected to compound‐specific stable isotope analysis. Turnover characteristics are calculated using a numerical advection–diffusion–decomposition model. Our measurements show significant differences in carbon storage (P<0.05) for both sites (Podocarpus 23.5 ± 3.2 kg SOC m^?3; Eucalyptus 18.6 ± 2.7 kg SOC m^?3). These differences can be explained with an initial loss of 15–26% of TOC about 50 years ago, induced by clearing the natural forest. After canopy closure, the carbon input below Eucalyptus is 15–34% less than below natural forest. At present, mean residence times (MRTs) of the investigated compounds do not differ between both stands. Sugars show the shortest MRTs in the topsoil with 2–7 years (xylose) and 5–13 years (arabinose) and have been affected the most by clear‐cutting. TOC and BC show MRTs of 13–25 years and 20–34 years, respectively. Old C4 carbon below 20 cm has merely been affected by the land use change. Contrary to expectation, our study does not indicate a pronounced recalcitrance of BC.     

Sequestration and turnover of plant- and microbially derived sugars in a temperate grassland soil during 7 years exposed to elevated atmospheric pCO2

Temperate grasslands contribute about 20% to the global terrestrial carbon (C) budget with sugars contributing 10–50% to this soil C pool. Whether the observed increase of the atmospheric CO₂ concentration (pCO₂) leads to additional C sequestration into these ecosystems or enhanced mineralization of soil organic matter (SOM) is still unclear. Therefore, the aim of the presented study was to investigate the impact of elevated atmospheric pCO₂ on C sequestration and turnover of plant‐ (arabinose and xylose) and microbially derived (fucose, rhamnose, galactose, mannose) sugars in soil, representing a labile SOM pool. The study was carried out at the Swiss Free Air Carbon Dioxide Enrichment (FACE) experiment near Zurich. For 7 years, Lolium perenne swards were exposed to ambient and elevated pCO₂ (36 and 60 Pa, respectively). The additional CO₂ in the FACE plots was depleted in ¹³C compared with ambient plots, so that ‘new’ (<7 years) C inputs could be determined by means of compound‐specific stable isotope analysis (¹³C : ¹²C). Samples were fractionated into clay, silt, fine sand and coarse sand, which yielded relatively stable and labile SOM pools with different turnover rates. Total sugar sequestration into bulk soil after 7 years of exposure to elevated pCO₂ was about 28% compared with the control plots. In both ambient and elevated plots, total sugar concentrations in particle size fractions increased in the order sand2 for coarse sand, fine sand and silt (about 274%, 17% and 96%, respectively) but about 14% lower for clay compared with the control plots, corroborating that sugars belong to the labile SOM pool. The fraction of newly produced sugars gradually increased by up to 50% in bulk soil samples after 7 years under elevated pCO₂. In the ambient plots, sugars were enriched in ¹³C by up to 10‰ when compared with bulk soil samples from the same plots. The enrichment of ¹³C in plant‐derived sugars was up to 13.4‰ when compared with parent plant material. After 7 years, the δ¹³C values of individual sugars decreased under elevated (¹³C‐depleted) CO₂ in bulk soil and particle size fractions, varying between −13.7‰ and −37.8‰ under elevated pCO₂. In coarse and fine sand, silt and clay fractions newly produced sugars made up 106%, 63%, 60% and 45%, respectively, of the total sugars present after 7 years. Mean residence time (MRT) of the sugars were calculated according to two models revealing a few decades, mean values increasing in the order coarse sand2 led to a net sequestration of about 30% of labile SOM (sugars) while no increase of total organic C was observed at the same plots. The additional labile SOM is gradually incorporated into more stable SOM pools such as silt and clay fractions in the medium term (<7 years). MRT of labile (sugar) SOM under elevated pCO₂ is in the same order of magnitude when compared with studies under ambient pCO₂ though no direct comparison of elevated and ambient plots was possible.