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Anaerobic oxidation of methane (AOM) is a globally important CH4 sink. However, the AOM pathways in paddy soils, the largest agricultural source of methane emissions (31 Mio tons per year) are not yet well described. Here, a combination of 13C isotope tracer, phospholipid fatty acids (PLFA) analyses, and microbial community analysis was used to identify AOM pathways in fertilized (pig manure, biochar, NPK, and the control) paddy soils amended with alternative electron acceptors (AEAs) (NO3 −, Fe3+, SO4 2−, humic acids, and the reference without AEAs addition). After 84 days of anaerobic incubation, the microbial co-occurrence network got tightened and became more complex relative to unincubated samples. Fertilization and AEAs addition led to a strong divergence of the microbial community structure as indicated by abundances of AOM-related microbiota and 13C incorporation into microbial PLFA, thus suggesting an environmental niche differentiation of AOM-involved microorganisms. Comparative analyses revealed a set of major and minor AOM pathways with synergistic relations to complementary anaerobic microbial groups. NO3 −-driven AOM, performed by members of the candidate group ANME-2d, was the major AOM pathway. Minor AOM pathways involved NO2 − reduction by NC10, reduction of humic acids and Fe3+ by Geobacter species, and SO4 2− reduction by sulfate-reducing bacteria linked with anaerobic methanotrophs. As identified by the network analysis, these active AOM pathways compensated a fraction of CH4 produced during ongoing methanogenesis. From a broader ecological perspective, nitrogen-driven AOM will become a more important methane sink in the future with the increases of nitrogen fertilization and deposition.
<p>Microbial transformation of organic substances is a key process of soil organic matter (SOM) formation. Carbon (C) entering the soil can be transformed in three main directions: i) stabilization over long period without relevant microbial utilization, ii) recycling by microorganisms for production of new and reparation of old cells, and iii) microbial utilization for energy production leading to C losses from soil as CO<sub>2</sub>. So, individual compounds within huge diversity of the organic substances entering the soil will follow predominantly one of these directions, depending on the substance chemistry, soil properties, microbial activities and environmental conditions. Therefore, organic substances can have two general trends: i) they converge from any initially distinct compounds (e.g. in litter or rhizodeposition) to completely mixed, so that it is impossible to trace back their origin; or ii) divergence: the substances maintain their differences despite microbial transformations by SOM formation.</p><p>We proved two opposite hypotheses that convergence and divergence of the fate of organic substances depends on microbial utilization at two levels: 1) intermolecular: high recycling intensity leads to convergence, whereas stabilization leads to divergence of the C originated from various organic compounds, and 2) incorporation of C from various molecule positions into microbial metabolic cycles define the C fate at intramolecular level. We tested the first hypothesis based on own and literature data to the fate of polymeric substances: sugars, proteins, lipids and lignin. The second hypothesis was tested by the C atoms from various positions of pentoses and hexoses by position-specific <sup>13</sup>C and <sup>14</sup>C labeling.</p><p>The polymeric substances as well as monomers from the same chemical group clearly converge to three groups stabilization, recycling and losses. Carboxylic acids will be nearly completely mineralized and are lost from soil. The fate and functions organic compounds depend mainly on microbial recycling. Proteins, amino acids and sugars - key components of microbial biomass - are intensively recycled and e.g. proteins remain relatively long in soil.</p><p>For the intramolecular differences, we traced the fate of position-specific <sup>13</sup>C labeled glucose and ribose under field conditions for 800 days. Both sugars were simultaneously metabolized via glycolysis and pentose phosphate pathway. The similarity between position-specific <sup>13</sup>C recovery in microbial biomass and soil reflected high contribution of microbial necromass to SOM. The mean residence time of uniformly labeled <sup>13</sup>C ribose in the soil was 3 times longer than that of glucose. Consequently, ribose and glucose were incorporated into different cellular components, defining their long-term fate in soil. The convergence of glucose C positions in soil and microbial biomass revealed that recycling dominated glucose transformation. In contrast, divergence of ribose C positions in soil revealed that intact ribose-derived cell components are reused or preserved in SOM.</p><p>Thus, convergence vs. divergence distinguished the two general trends explaining the long persistence of C at inter- and intra-molecular levels: microbial recycling leads to convergence, whereas slow decomposition and preservation define the divergence of C pathways in soil.</p>
Tropical mountain ecosystems cover a broad variety of climatic and vegetation zones and are global hotspots of biodiversity. These ecosystems are severely threatened by climate and land-use change, which also strongly affect soil properties. Mt. Kilimanjaro, with its large elevation gradient and relatively homogeneous geology of volcanic rocks and ashes, provides a unique opportunity to study and interpret soil organic matter (SOM) responses to climatic changes. Our objectives were to identify key SOM compounds in six elevation zones (covering ecosystems from tropical dry-lowland, through montane forest, up to alpine heathlands) that are affected by the climatic changes along a 3400 m transect, and to relate these SOM changes to ecosystem specific characteristics. The SOM composition in four topsoils (0–10 cm) samples per ecosystem was characterized by analytical double-shot pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS). Evolving-gas analysis-mass spectrometry (EGA-MS) was used to quantitatively compare the thermal desorption (first shot: <280 °C) and pyrolysis step (second shot: 280–600 °C). The percentage of thermally desorbed compounds increased 10 fold from lowland (<900 m) to mountain forest soils (>2000 m), followed by a 40% decrease in alpine ecosystems (>4000 m). Alkanes/-enes/-ols contributed between 4% and 30% to the identified SOM composition, with a maximum at mid elevation (2120 m). Fatty acids and fatty acid esters contributed with <3% to SOM composition and decreased to a minimum of <1% at Podocarpus forest soils (2900 m), followed by a re-increase at higher elevation. The percentage of lignin-derived compounds followed a similar pattern but also responded to reduced woody inputs above the tree line. Two main factors that seem to affect SOM quality and composition at Mt. Kilimanjaro were: 1) the rate and composition of organic matter inputs that in turn are controlled by climatic characteristics and the vegetation type and 2) the decomposition rate and efficiency, mainly controlled by soil pH, temperature and water availability. High forest productivity at mid elevations (2200 m) leads to high amounts of volatile compounds and increases stable SOM pools. The overall carbon accumulation in Andosols of Mt. Kilimanjaro is linked to the percentage of bound lipids (mainly alkanes, alkenes and alcohols), while site specific input patterns (e.g. vegetation or wild fires) are strongly reflected by sterols, lignin derived compounds or polycyclic aromatic compounds in SOM.
Climate change projections indicate that significant areas of the current cocoa cultivation areas in West Africa are likely to experience unfavorable climatic conditions by 2050. Water use efficient agroforestry systems are considered to be an important option to adapt cocoa to climate change. Water use efficiency and complementary soil water use between cocoa and shade trees have been reported in previous studies in Indonesia but in our previous study in Ghana, popular native shade tree species Albizia ferruginea and Antiaris toxicaria were found to have a strong competitive water use advantage over cocoa plants during an extreme drought experienced during 2015/16. Cocoa plants under no shade were relatively more resilient with higher survival rate and post drought recovery.While past research studies &#160;&#160;were each limited to few selected shade tree species being investigated despite the huge numbers within cocoa landscapes. To overcome this limitation, an approach based on functional traits of trees in terms of water use is proposed to understand their effect on water use efficiency and drought resilience in cocoa agroforestry systems rather than describing individual species. We apply this concept across temporal and spatial scales in a marginally suitable cocoa climate in Ghana. Shade tree species have been categorized into phenological trait groups (evergreen, deciduous, or brevi-deciduous) under which detailed above- and belowground traits interactions with cocoa plant and effect on water use has been evaluated.The following hypotheses are being tested: (i) shade trees in the three phenological trait groups exhibit significant difference in their root and water uptake depth, and, thus, affect environmental conditions relevant for cocoa, (ii) cocoa plant above- and belowground morphological and physiological traits are influenced by shade tree phenological and morphological traits and their modification by micro-climatic (light, temperature, relative humidity and VPD), and soil (water and nutrients) conditions over different seasons.Replicated plots of 19 shade tree species distributed across the three phenological trait groups have been established for analysis of functional traits interactions in such a multi-species agroforestry system. We determine root specific traits through direct sampling, stable isotope analysis for assessment of water use portioning between the various shade tree groups and cocoa plants. Soil moisture, temperature and relative humidity and light sensors were installed in each plot. Cocoa plants under different micro-climatic impact zones of the shade trees have been monitored for a full production cycle. &#160;The shade trees impact on the cocoa plant productivity (morphology and yield traits) has been evaluated. Complementary and non-complementary shade tree species with respect to their trait interactions and effect on cocoa plant productivity have been identified.
&lt;p&gt;Biogeochemical cycles of phosphorus (P) and iron (Fe) are tightly intertwined, especially in highly weathered and acidic subtropical and tropical soils rich in ferric Fe (Fe(III))oxides. In low-redox and P-deficient paddy soils, the quantitative contribution of the reductive dissolution of Fe(III)-bound P (Fe-P) to the demands of rice plants (&lt;em&gt;Oryza sativa&lt;/em&gt;&amp;#160;L.) and microorganisms remains unclear.&amp;#160;We hypothesized that Fe(III) reductive dissolution can cover the P demand of microorganisms but not of rice plants during the initial growth stages, when P demand is high but the root system is still limited.&amp;#160;We grew pre-germinated rice plants for 33 days in flooded rhizoboxes filled with a paddy soil&amp;#160;of poor P availability. &lt;sup&gt;32&lt;/sup&gt;P-labeled orthophosphate sorbed to ferrihydrite (80 kg ha&lt;sup&gt;-1&lt;/sup&gt;) was supplied either&amp;#160;(1) in&amp;#160;polyamide mesh bags (30 &amp;#956;m mesh size) to prevent roots from directly mobilizing Fe-P&amp;#160;(Pellets-mesh bag treatment), or&amp;#160;(2)&amp;#160;in the form of pellets directly to the soil without mesh bags to enable roots&amp;#8217; accessing the&amp;#160;Fe-P (Pellets-no-mesh bag&amp;#160;treatment). With&amp;#160;the application of Fe-P directly to the soil,&amp;#160;P was more available resulting in the increases in microbial biomass carbon (MBC) by 18&amp;#8211;55% and&amp;#160;nitrogen (MBN) by 4&amp;#8211;108% in rooted soil as&amp;#160;compared to the pellet not available to roots directly.&amp;#160;The maximum enzyme activities (V&lt;sub&gt;max&lt;/sub&gt;) of phosphomonoesterase and &amp;#946;-glucosidase followed this pattern.&amp;#160;During rice root growth, MBC&amp;#160;and&amp;#160;microbial biomass phosphorus (MBP) in both rooted and bottom bulk soil&amp;#160;gradually decreased by 28&amp;#8211;56% and&amp;#160;47&amp;#8211;49%, respectively. In contrast to our hypothesis, the contribution of Fe-P to MBP strongly decreased from 4.5% to almost zero during 10&amp;#8211;33 days after rice transplantation, while&amp;#160;Fe-P compensated up to 16% of the plant P&amp;#160;uptake 33 days after rice transplantation, thus outcompeting microorganisms.&lt;/p&gt;
The anaerobic oxidation of methane (AOM) in marine ecosystems is ubiquitous and largely coupled to sulfate reduction. In contrast, the role of AOM in terrestrial environments and the dominant electron acceptors driving terrestrial AOM needs deeper understanding. Submerged rice paddies with intensive CH4 production have a high potential for AOM, which can be important for greenhouse gas mitigation strategies. Here, we used 13CH4 to quantify the AOM rates in paddy soils under organic (Pig manure, Biochar) and mineral (NPK) fertilization. Alternative-to-oxygen electron acceptors for CH4 oxidation, including Fe3+, NO3 −, SO4 2−, and humic acids, were examined and their potential for CH4 mitigation from rice paddies was assessed by 13CH4 oxidation to 13CO2 under anoxic conditions. During 84 days of anaerobic incubation, the cumulative AOM (13CH4-derived CO2) reached 0.15–1.3 μg C g-1 dry soil depending on fertilization. NO3- was the most effective electron acceptor, yielding an AOM rate of 0.80 ng C g-1 dry soil h-1 under Pig manure. The role of Fe3+ in AOM remained unclear, whereas SO42- inhibited AOM but strongly stimulated the production of unlabeled CO2, indicating intensive sulfate-induced decomposition of organic matter. Humic acids were the second most effective electron acceptor for AOM, but increased methanogenesis by 5–6 times in all fertilization treatments. We demonstrated for the first time that organic electron acceptors (humic acids) are among the key AOM drivers and are crucial in paddy soils. The most pronounced AOM in paddy soils occurred under Pig manure, followed by Control and NPK, while AOM was the lowest under Biochar. We estimate that nitrate (nitrite)-dependent AOM in paddy fields globally consumes ~3.9 Tg C–CH4 yr-1, thereby offsetting the global CH4 emissions by ~10–20%. Thus, from a broader agroecological perspective, the organic and mineral fertilizers control an important CH4 sink under anaerobic conditions in submerged ecosystems. Appropriate adjustments of soil fertilization management strategies would therefore help to decrease the net CH4 flux to the atmosphere and hence the global warming.
When multiple metabolic pathways lead to the same product, compound-specific isotope analysis may not provide enough information to quantify the activities of the contributing pathways. Instead, identification of where in the molecule the 13C is incorporated is required. Here we show how knowledge of position-specific 13C incorporation in fatty acids (FA) and FA fragments can be used to quantitatively estimate the fluxes through the central C metabolic network. We developed a method to measure 13C enrichment of FA and FA fragments (ethanoate, propionate) using electron impact GC–MS. We tested the accuracy and repeatability of the measurements using natural abundance and position-specific 13C labelled standards and FA extracted from Bacillus licheniformis and Pseudomonas fluorescens grown with labelled and unlabelled glucose. The molecular ions of FA generally reflected theoretical predictions of mass isotopomer distributions for natural abundance values, but that of the associated FA fragments deviated from expected values, likely associated with McLafferty rearrangements of hydrogen. After correction for naturally occurring isotopes, 13C enrichments of FA and FA fragments showed good agreement with expected isotope composition of FA standards (root mean square error < 0.044 at%; δ13C of ∼ 40‰), natural abundance and labelled glucose. The unsaturated FA extracted from P. fluorescens deviated from expected values likely associated with problems of co-elution and ion suppression and were excluded from analysis. The ratio of glucose-1-13C to glucose-3-13C incorporation into FA fragments was high for B. licheniformis, but low for P. fluorescens. Metabolic flux modelling based on the 13C enrichment of ethanoate and propionate fragments showed that B. licheniformis used Embden-Meyerhof-Parnas and pentose phosphate pathway (66% and 30%, respectively), whereas P. fluorescens utilized Entner-Doudoroff and pentose phosphate pathway (72% and 27%, respectively). FA fragment analysis is therefore a promising tool to study central C metabolic network activities of co-occurring groups of microbes in intact and complex environmental communities.
Psychrotolerant microbes are crucial for carbon cycling and biotechnological applications. Nonetheless, the mechanisms enabling their survival and function
. Independent of its chemical structure carbon (C) persists in soil for several decades, controlled by stabilisation and recycling. To disentangle the importance of the two factors on the turnover dynamics of soil sugars, an important compound of soil organic matter (SOM), a three year incubation experiment was conducted on a silty loam soil under different types of land use (arable land, grassland and forest) by adding 13C-labeled glucose. The compound specific isotope analysis of soil sugars was used to examine the dynamics of different sugars during incubation. Sugar dynamics were dominated by a pool of high mean residence times (MRT) indicating that recycling plays an important role for sugars. However, this was not substantially affected by soil C content. Six months after label addition the contribution of the label was much higher for microbial biomass than for CO2 production for all examined soils, corroborating that substrate recycling was very effective within the microbial biomass. Two different patterns of tracer dynamics could be identified for different sugars: while fucose (fuc) and mannose (man) showed highest label contribution at the beginning of the incubation with a subsequent slow decline, galactose (gal) and rhamnose (rha) were characterised by slow label incorporation with subsequently constant levels, which indicates that recycling is dominating the dynamics of these sugars. This may correspond to (a) different microbial growing strategies (r and K-strategist) or (b) location within or outside the cell membrane (lipopolysaccharides vs. exopolysaccharides) and thus be subject of different re-use within the microbial food web. Our results show how the microbial community recycles substrate very effectively and that high losses of substrate only occur during initial stages after substrate addition.
Understanding the pathways of nitrogen (N) retention in pristine forest soils is essential for effective ecosystem management and nutrient conservation. The incorporation of nitrate (NO<sub>3</sub><sup>-</sup>) and nitrite (NO<sub>2</sub><sup>-</sup>) into organic N in soils without microbiological contribution remains a very intriguing question. This study explores the abiotic incorporation of nitrate (NO<sub>3</sub><sup>-</sup>) and nitrite (NO<sub>2</sub><sup>-</sup>) into organic N of volcanic soil under sterilized and anoxic conditions, providing insights into mineral N losses occurring as dissolved organic N (DON) rather than the commonly accepted nitrate leaching. We evaluated the hypothesis that nitrate (NO<sub>3</sub><sup>-</sup>) can be reduced to nitrite (NO<sub>2</sub><sup>-</sup>), which subsequently reacts with organic matter through nitration and nitrosation, leading to the formation of organic nitrogen. This mechanism, which is of great ecological significance, supports the Ferrous Wheel Hypothesis (FWH). The FWH proposes that ferrous iron, Fe(II), reduces NO<sub>3</sub><sup>-</sup> to NO<sub>2</sub><sup>-</sup> within anaerobic microsites, and that Fe(II) is then re-oxidised to ferric iron, Fe(III), contributing to the formation of dissolved organic N (DON). Both NO<sub>3</sub><sup>-</sup> and NO<sub>2</sub><sup>-</sup> declined rapidly by 51 and 94 %, while labelled organic N increased by 20-38 % for NO<sub>3</sub><sup>-</sup> and 42-44 % for NO<sub>2</sub><sup>-</sup> within seconds. The incorporation of <sup>15</sup>N into organic forms was confirmed using ATR-FTIR and benzene:isopropanol extraction, with the lowest and highest accumulation observed at 5 and 15 mg NO<sub>₃</sub>⁻ kg⁻¹, respectively. These results demonstrate that NO<sub>3</sub><sup>-</sup> incorporation into organic N can occur primarily through abiotic processes, supporting the FWH, as both DON and solid-phase organic N were measured. These findings highlight the natural resilience of volcanic soils in unpolluted old-growth temperate rainforests to N loss and provide new insights into long-term ecosystem stability and nutrient cycling. Further research should investigate the interplay between abiotic and biotic N transformations under field conditions and across diverse forest ecosystems.
