1,721,177 research outputs found
Direct incorporation of fatty acids into microbial phospholipids in soils: Position-specific labeling tells the story
No full textFatty acids have been used as plant and microbial biomarkers, and knowledge about their transformation pathways in soils and sediments is crucial for interpreting fatty acid signatures, especially because the formation, recycling and decomposition processes are concurrent. We analyzed the incorporation of free fatty acids into microbial fatty acids in soil by coupling position-specific C-13 labeling with compound-specific C-13 analysis. Position-specifically and uniformly C-13 labeled palmitate were applied in an agricultural Luvisol. Pathways of fatty acids were traced by analyzing microbial utilization of C-13 from individual molecule positions of palmitate and their incorporation into phospholipid fatty acids (PLFA). The fate of palmitate C-13 in the soil was characterized by the main pathways of microbial fatty acid metabolism: Odd positions (C-1) were preferentially oxidized to CO2 in the citric acid cycle, whereas even positions (C-2) were preferentially incorporated into microbial biomass. This pattern is a result of palmitate cleavage to acetyl-CoA and its further use in the main pathways of C metabolism. We observed a direct, intact incorporation of more than 4% of the added palmitate into the PLFA of microbial cell membranes, indicating the important role of palmitate as direct precursor for microbial fatty acids. Palmitate C-13 was incorporated into PLFA as intact alkyl chain, i.e. the C backbone of palmitate was not cleaved, but palmitate was incorporated either intact or modified (e.g. desaturated, elongated or branched) according to the fatty acid demand of the microbial community. These modifications of the incorporated palmitate increased with time. Future PLFA studies must therefore consider the recycling of existing plant and microbial-derived fatty acids. This study demonstrates the intact uptake and recycling of free fatty acids such as palmitate in soils, as well as the high turnover and transformation of cellular PLFA. Knowledge about the intact uptake and use of soil-derived free fatty acids is crucial for interpreting microbial fatty acid fingerprints and their isotopic composition. (c) 2015 Elsevier Ltd. All rights reserved.Deutsche Forschungsgemeinschaft (DFG) [KU1184/19-1, DI2136/1-1
Biogeochemical transformations of amino acids in soil assessed by position-specific labelling
No full textAmino acid turnover in soil is an important element of terrestrial carbon and nitrogen cycles. This study accounts for their driver - the microbial metabolism - by tracing them via the unique isotopic approach of position-specific labeling. Three C-14 isotopomers of alanine at five concentration levels combined with selective sterilization were used to distinguish sorption mechanisms, exoenzymatic and microbial utilization of amino acids in soil. Sorption and microbial uptake occurred immediately. Unspecific microbial uptake followed a linear kinetic, whereas energy-dependent uptake followed Michaelis-Menten. Less than 6 % of the initially added alanine was sorbed to soil, but after microbial transformation products were bound to the soil matrix at higher proportions (5-25 %). The carboxyl group (C-1) was rapidly oxidized by microorganisms, whereas C-2 and C-3 positions were preferentially incorporated into microbial biomass. Dependency of C metabolization on amino acid concentration reflected individual alanine transformation pathways for starvation, maintenance and growth conditions. This study demonstrates that position-specific labeling determines the mechanisms and rates of C cycling from individual functional groups. This approach reflected underlying metabolic pathways and revealed the formation of new organic matter. We therefore conclude that position-specific labeling is a unique tool for detailed insights into submolecular transformation pathways and their regulation factors.Deutsche Forschungsgemeinschaft (DFG
13C analysis of fatty acid fragments by gas chromatography mass spectrometry for metabolic flux analysis
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Sorption affects amino acid pathways in soil: Implications from position-specific labeling of alanine
No full textOrgano-mineral interactions are the most important mechanisms of long-term C stabilization in soils. Nevertheless, a part of the sorbed low molecular weight organic substances (LMWOS) remains bioavailable. Uniformly labeling of substances by C-14 or C-13 reflects only the average fate of C atoms of a LMWOS molecule. The submolecular tool of position-specific labeling allows to analyze metabolic pathways of individual functional groups and thus reveals deeper insight into mechanisms of sorption and microbial utilization. Alanine labeled with C-14 in the 1st, 2nd or 3rd position was adsorbed to five sorbents: two iron oxides with different crystalline structure: goethite and haematite; two clay minerals with 2:1 layers smectite, and 1:1 layers kaolinite; and activated charcoal. After subsequent addition of these sorbents to a loamy haplic Luvisol, we analyzed C-14 release into the soil solution, its microbial utilization and (CO2)-C-14 efflux from individual C positions of alanine. All sorbents bound alanine as an intact molecule (identical sorption of 1st, 2nd or 3rd positions). The bioavailability of sorbed alanine and its microbial transformation pathways depended strongly on the sorbent. Goethite and activated charcoal sorbed the highest amount of alanine (similar to 45% of the input), and the lowest portion of the sorbed alanine C was microbially utilized (26 and 22%, respectively). Mineralization of the desorbed alanine peaked within the first 5 h and was most pronounced for alanine bound to clay minerals. The initial mineralization to CO2 of bound alanine was always highest for the C-1 position (-COOH group). Mineralization rates of C-2 and C-3 exceeded the C-1 oxidation after 10-50 h, reflecting the classical biochemical pathways: 1) deamination, 2) decarboxylation of C-1 within glycolysis, and further 3) oxidation of C-2 and C-3 in the citric acid cycle. The ratio between two metabolic pathways glycolysis (C-1 oxidation) versus citric-acid cycle (oxidation of C-2 and C-3) was dependent on the microbial availability of sorbed alanine. High availability causes a peak in glycolysis C-1 oxidation followed by an abrupt shift to oxidation via the citric acid cycle. Low microbial availability of sorbed alanine, in turn, leads to a less pronounced, parallel oxidation of all three positions and to a higher relative incorporation of alanine C into microbial compounds. Modeling of C fluxes revealed that a significant portion of the sorbed alanine was incorporated in microbial biomass after 78 h and was further stabilized at the sorbents' surfaces. Position-specific labeling enabled determination of pathways and rates of C utilization from individual molecule positions and its dependence on various sorption mechanisms. We conclude that position-specific labeling is a unique tool for detailed insights into the submolecular transformation processes, mechanisms and rates of C stabilization in soil. (C) 2014 Elsevier Ltd. All rights reserved.Deutsche Forschungsgemeinschaf
Manufacturing triple-isotopically labeled microbial necromass to track C, N and P cycles in terrestrial ecosystems
No full textThe functional relevance of microbial necromass in terrestrial biogeochemical cycles remains one of the unresolved mysteries of element cycling in ecosystems, especially considering the high microbial abundance and turnover in soil. We therefore established a protocol to manufacture multi-isotope (14C, 15N and 33P) labeled microbial necromass to comprehensively track the turnover of microbial necromass elements within element cycles. This protocol encompasses the i) microbial cultivation of Pseudomonas kilonensis ACN4 (Gram-negative) and Bacillus licheniformis DSM13 (Gram-positive) on labeled minimal medium as well as fungal cultivation of Hypsizygus tessulatus on a complex yeast medium, ii) quantification of radio- (14C, 33P) and stable (15N) isotope incorporation as well their cellular pool partitioning, and iii) determination of element and tracer isotope uptake efficiency. We achieved 1 g of bacterial biomass per liter minimum medium within 24 h and 2.9 g l-1 fungal biomass in complex medium within 18 d. This production rate enabled us to produce more than 100 g of necromass within only one half-life time of 33P, including post-harvest processing. Isotope uptake and incorporation for 33P ranged from 10 to 73%, for 15N from 24 to 52%, and for 14C from 12 to 23%. Each of the cultivated species showed individual patterns of tracer element uptake. The nutritional value of the carbon- (C), nitrogen- (N) and phosphorus- (P) labeled microbial necromass was characterized by a water-based, necromass speciesspecific partitioning scheme with subsequent elemental analysis of the pools. We separated Gram-negative, Gram-positive and fungi’s cellular pools to characterize element and tracer partitioning among dissolved versus particulate fractions. That is essential because these properties subsequently affect the respective pool's availability for ecosystem nutrition. Our procedure allows a defined production of microorganism-based necromass, enabling versatile use to determine necromass-related nutrient fluxes in terrestrial ecosystem studies
Allocation and dynamics of C and N within plant-soil system of ash and beech
No full textForest management requires a profound understanding of how tree species affect C and N cycles in ecosystems. The large C and N stocks in forest soils complicate research on the effects of tree species on C and N pools. In-situ C-13 and N-15 labeling in undisturbed, natural forests enable not only tracing of C and N fluxes, but also reveal insight into the interactions at the plant-soil-atmosphere interface. In-situ dual C-13 and N-15 pulse labeling of 20 beeches (Fagus sylvatica L.) and 20 ashes (Fraxinus excelsior L.) allowed tracing the fate of assimilated C and N in trees and soils in an unmanaged forest system in the Hainich National Park (Germany). Leaf, stem, root, and soil samples as well as microbial biomass were analyzed to quantify the allocation of 13C and N-15 for 60 d after labeling and along spatial gradients in the soil with increasing distance from the stem. For trees of similar heights (approximate to 4 m), beech (20%) assimilated twice as much as ash (9%) of the applied (CO2)-C-13, but beech and ash incorporated similar N-15 amounts (45%) into leaves. The photosynthates were transported belowground through the phloem more rapidly in beech than in ash. Ash preferentially accumulated N-15 and C-13 in the roots. In contrast, beech released more of this initially assimilated C-13 (2.0% relative C-13 allocation) and N-15 (0.1% relative N-15 allocation) via rhizodeposition into the soil than ash (0.2% relative C-13, 0.04% relative N-15 allocation), which was also subsequently recovered in microbial biomass. These results on C and N partitioning contribute to an improved understanding of the effects of European beech and ash on the C and N cycles in deciduous broad-leaved forest. Differences in C and N allocation patterns between ash and beech are one mechanism of niche differentiation in forests containing both species.German Research Foundation (DFG); DFG Graduiertenkolleg 108
Sorption of Alanine changes microbial metabolism in addition to availability
No full textSorption is one of the main processes stabilizing organic matter in soil against microbial mineralization. We hypothesize that besides reduced accessibility for microorganisms and enzymes, changes in microbial metabolism additionally intensify this organic matter stabilization effect of sorption. Position-specifically C-14 labeled Alanine was applied to soil as solution or sorbed on sterilized soil to investigate the mechanisms underlying this metabolism related stabilization effect. Sorption decreased initial mineralization of Alanine by-80% and doubled the duration until the mineralization maxima ((CO2)-C-14 peak). Almost all Alanine was taken up by microorganisms independent on sorption, and C-1 was completely (>99%) decarboxylated during glycolysis after one day. Sorption could not prevent microbial utilization of Alanine, but increased the carbon use efficiency (CUE) of sorbed Alanine for 60% compared to Alanine in solution and increased C incorporation in microbial biomass up to four times. The position-specific pattern of C-14 in soil and in microbial biomass showed that oxidation of C-2 from sorbed Alanine was strongly lowered compared to free Alanine. Both higher CUE and delayed C-2 mineralization were achieved by a higher C flux towards efficient anabolism, or/and to slower cycling cell components. Limitation of accessibility for microorganisms alone does not explain the stabilizing effect of sorption on organic substances like amino acids and the observed changed position specific pattern. Even though all sorbed Alanine was taken up by microorganisms within 3 days, C partitioning towards anabolism, slower microbial turnover and increased CUE increased C retention from sorbed compounds in soil even after microbial uptake. Position-specific labeling clearly showed that LMWOS are stabilized by sorption not as intact molecules, but after microbial metabolization- as released metabolites or microbial biomass. We conclude that the indirect effects of sorption, namely 1) more C partitioned to anabolism, 2) slower decomposition, 3) higher incorporation into microbial biomass and 4) increased carbon use efficiency promote C retention in soil and may be even more important than the direct effect, namely inaccessibility. The finding that stabilization did not significantly impede microbial utilization, but sorption greatly increased carbon use efficiency has major implications for conceptual and numerical representation of organic matter stabilization and losses in soils. (C) 2017 Elsevier B.V. All rights reserved.Deutsche Bundesstiftung Umwelt; DFG [DI2136 1/1]; INST [186/1006-1 FUGG
Keep oxygen in check: Contrasting effects of short-term aeration on hydrolytic versus oxidative enzymes in paddy soils
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