Michaela A. Dippold

Biochar affects soil organic matter cycling and microbial functions but does not alter microbial community structure in a paddy soil

The application of biochar (BC) in conjunction with mineral fertilizers is one of the most promising management practices recommended to improve soil quality. However, the interactive mechanisms of BC and mineral fertilizer addition affecting microbial communities and functions associated with soil organic matter (SOM) cycling are poorly understood. We investigated the SOM in physical and chemical fractions, microbial community structure (using phospholipid fatty acid analysis, PLFA) and functions (by analyzing enzymes involved in C and N cycling and Biolog) in a 6-year field experiment with BC and NPK amendment. BC application increased total soil C and particulate organic C for 47.4-50.4% and 63.7-74.6%, respectively. The effects of BC on the microbial community and C-cycling enzymes were dependent on fertilization. Addition of BC alone did not change the microbial community compared with the control, but altered the microbial community structure in conjunction with NPK fertilization. SOM fractions accounted for 55% of the variance in the PLFA-related microbial community structure. The particulate organic N explained the largest variation in the microbial community structure. Microbial metabolic activity strongly increased after BC addition, particularly the utilization of amino acids and amines due to an increase in the activity of proteolytic (l-leucine aminopeptidase) enzymes. These results indicate that microorganisms start to mine N from the SOM to compensate for high C:N ratios after BC application, which consequently accelerate cycling of stable N. Concluding, BC in combination with NPK fertilizer application strongly affected microbial community composition and functions, which consequently influenced SOM cycling.

Combined quantification of faecal sterols, stanols, stanones and bile acids in soils and terrestrial sediments by gas chromatography-mass spectrometry.

Faeces incorporation can alter the concentration patterns of stanols, stanones, Δ(5)-sterols and bile acids in soils and terrestrial sediments. A joint quantification of these substances would give robust and specific information about the faecal input. Therefore, a method was developed for their purification and determination via gas chromatography-mass spectrometry (GC-MS) based on a total lipid extract (TLE) of soils and terrestrial sediments. Stanols, stanones, Δ(5)-steroles and bile acids were extracted by a single Soxhlet extraction yielding a TLE. The TLE was saponified with KOH in methanol. Sequential liquid-liquid extraction was applied to recover the biomarkers from the saponified extract and to separate the bile acids from the neutral stanoles, stanones and Δ(5)-steroles. The neutral fraction was directly purified using solid phase extraction (SPE) columns packed with 5% deactivated silica gel. The bile acids were methylated in dry HCl in methanol and purified on SPE columns packed with activated silica gel. A mixture of hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS) and pyridine was used to silylate the hydroxyl groups of the stanols and Δ(5)-sterols avoiding a silylation of the keto groups of the stanones in their enol-form. Silylation of the bile acids was carried out with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing N-trimethylsilylimidazole (TSIM). TLEs from a set of soils with different physico-chemical properties were used for method evaluation and for comparison of amounts of faecal biomarkers analysed with saponification and without saponification of the TLE. Therefore, a Regosol, a Podzol and a Ferralsol were sampled. To proof the applicability of the method for faecal biomarker analyses in archaeological soils and sediments, additional samples were taken from pre-Columbian Anthrosols in Amazonia and an Anthrosol from a site in central Europe settled since the Neolithic. The comparison of the amounts of steroids in combination with and without saponification of the TLE showed that high amounts of faecal biomarkers occur bound to other lipids and were liberated by saponification. The method was evaluated by standard addition. The standard contained 5β-stanols, 5β-stanones and their 5α-isomers together with Δ(5)-sterols and bile acids (19 substances). The standard addition revealed mean recoveries of individual substances ≥85%. The recoveries of biomarkers within each biomarker group did not differ significantly. Precisions were ≤0.22 (RSD) and quantification limits were between 1.3 and 10 ng g(-1) soil. These data showed that the method can be applied for quantification of trace amounts of faecal steroids and for the analyses of steroid patterns to detect enhanced faeces deposition in soils and sediments.

Amino acid fingerprint of a grassland soil reflects changes in plant species richness

A positive plant diversity to plant aboveground productivity relation has been shown to alter carbon and nitrogen fluxes in soils. Thus, most investigations focussed on the C- and N-input via litter fall, widely neglecting the importance of root exudation. As microbes, which are known to be important drivers of matter fluxes in soil, feed on these root exudates, increased knowledge on the availability of these compounds in soil might help to understand biodiversity effects on soil. We therefore investigated the effect of plant diversity on size and composition of the free soil amino acid pool in a grassland experiment, as amino acids are an important C- as well as N-source for microbes. Despite a positive diversity effect on plant productivity, we only found an insignificant increase of the size of the free amino acid pool in soil. This was most likely caused by an increase of the microbial population and thus an increased amino acid mineralization. At the same time the composition of the amino acid pool changed significantly between plant diversity levels. This most likely reflects differences in plant input as well as differences in microbial mineralization and enabled us to separate diversity levels by means of discriminant analysis.

Disentangling interactions between microbial communities and roots in deep subsoil

Soils, paleosols and terrestrial sediments serve as archives for studying climate change, and represent important terrestrial carbon pools. Archive functioning relies on the chronological integrity of the respective units. Incorporation of younger organic matter (OM) e.g. by plant roots and associated microorganisms into deep subsoil and underlying soil parent material may reduce reliability of paleoenvironmental records and stability of buried OM. Long-term effects of sedimentary characteristics and deep rooting on deep subsoil microbial communities remain largely unknown. We characterized fossil and living microbial communities based on molecular markers in a Central European Late Pleistocene loess-paleosol sequence containing recent and ancient roots with ages of several millenia. The molecular approach, comprising free and phospholipid fatty acids (FAs), core and intact polar glycerol dialkyl glycerol tetraethers (GDGTs), as well as 16S rRNA genes from bacterial DNA, revealed the presence of living microorganisms along the sequence, with bacterial community composition comparable to that of modern topsoils. Up to 88% redundancy between bacterial genetic fingerprint and molecular signature of fossil microorganisms suggested a time-integrated signal of the molecular markers accumulated over a time span potentially lasting from sedimentation over one or more rooting phases until today. Free FAs, core GDGTs and DNA, considered as remains of fossil microorganisms, corresponded with ancient and recent root quantities, whereas phospholipid FAs and intact polar GDGTs, presumably derived from living microorganisms, correlated only with living roots. The biogeochemical and ecological disequilibrium induced by postsedimentary rooting may entail long-term microbial processes like OM mineralization, which may continue even millenia after the lifetime of the root. Deep roots and their fossil remains have been observed in various terrestrial settings, and roots as well as associated microorganisms cause both, OM incorporation and mineralization. Therefore, these findings are crucial for improved understanding of OM dynamics and carbon sequestration potential in deep subsoils.

Allocation of freshly assimilated carbon into primary and secondary metabolites after in situ 13C pulse labelling of Norway spruce (Picea abies)

Plants allocate carbon (C) to sink tissues depending on phenological, physiological or environmental factors. We still have little knowledge on C partitioning into various cellular compounds and metabolic pathways at various ecophysiological stages. We used compound-specific stable isotope analysis to investigate C partitioning of freshly assimilated C into tree compartments (needles, branches and stem) as well as into needle water-soluble organic C (WSOC), non-hydrolysable structural organic C (stOC) and individual chemical compound classes (amino acids, hemicellulose sugars, fatty acids and alkanes) of Norway spruce (Picea abies) following in situ (13)C pulse labelling 15 days after bud break. The (13)C allocation within the above-ground tree biomass demonstrated needles as a major C sink, accounting for 86% of the freshly assimilated C 6 h after labelling. In needles, the highest allocation occurred not only into the WSOC pool (44.1% of recovered needle (13)C) but also into stOC (33.9%). Needle growth, however, also caused high (13)C allocation into pathways not involved in the formation of structural compounds: (i) pathways in secondary metabolism, (ii) C-1 metabolism and (iii) amino acid synthesis from photorespiration. These pathways could be identified by a high (13)C enrichment of their key amino acids. In addition, (13)C was strongly allocated into the n-alkyl lipid fraction (0.3% of recovered (13)C), whereby (13)C allocation into cellular and cuticular exceeded that of epicuticular fatty acids. (13)C allocation decreased along the lipid transformation and translocation pathways: the allocation was highest for precursor fatty acids, lower for elongated fatty acids and lowest for the decarbonylated n-alkanes. The combination of (13)C pulse labelling with compound-specific (13)C analysis of key metabolites enabled tracing relevant C allocation pathways under field conditions. Besides the primary metabolism synthesizing structural cell compounds, a complex network of pathways consumed the assimilated (13)C and kept most of the assimilated C in the growing needles.

Improved δ13C analysis of amino sugars in soil by ion chromatography–oxidation–isotope ratio mass spectrometry

RATIONALE Amino sugars build up microbial cell walls and are important components of soil organic matter. To evaluate their sources and turnover, δ(13)C analysis of soil-derived amino sugars by liquid chromatography was recently suggested. However, amino sugar δ(13)C determination remains challenging due to (1) a strong matrix effect, (2) CO2 -binding by alkaline eluents, and (3) strongly different chromatographic behavior and concentrations of basic and acidic amino sugars. To overcome these difficulties we established an ion chromatography-oxidation-isotope ratio mass spectrometry method to improve and facilitate soil amino sugar analysis. METHODS After acid hydrolysis of soil samples, the extract was purified from salts and other components impeding chromatographic resolution. The amino sugar concentrations and δ(13)C values were determined by coupling an ion chromatograph to an isotope ratio mass spectrometer. The accuracy and precision of quantification and δ(13)C determination were assessed. RESULTS Internal standards enabled correction for losses during analysis, with a relative standard deviation <6%. The higher magnitude peaks of basic than of acidic amino sugars required an amount-dependent correction of δ(13)C values. This correction improved the accuracy of the determination of δ(13)C values to <1.5‰ and the precision to <0.5‰ for basic and acidic amino sugars in a single run. CONCLUSIONS This method enables parallel quantification and δ(13)C determination of basic and acidic amino sugars in a single chromatogram due to the advantages of coupling an ion chromatograph to the isotope ratio mass spectrometer. Small adjustments of sample amount and injection volume are necessary to optimize precision and accuracy for individual soils.

Microbial Metabolism in Soil at Subzero Temperatures: Adaptation Mechanisms Revealed by Position-Specific 13C Labeling

Although biogeochemical models designed to simulate carbon (C) and nitrogen (N) dynamics in high-latitude ecosystems incorporate extracellular parameters, molecular and biochemical adaptations of microorganisms to freezing remain unclear. This knowledge gap hampers estimations of the C balance and ecosystem feedback in high-latitude regions. To analyze microbial metabolism at subzero temperatures, soils were incubated with isotopomers of position-specifically 13C-labeled glucose at three temperatures: +5 (control), -5, and -20°C. 13C was quantified in CO2, bulk soil, microbial biomass, and dissolved organic carbon (DOC) after 1, 3, and 10 days and also after 30 days for samples at -20°C. Compared to +5°C, CO2 decreased 3- and 10-fold at -5 and -20°C, respectively. High 13C recovery in CO2 from the C-1 position indicates dominance of the pentose phosphate pathway at +5°C. In contrast, increased oxidation of the C-4 position at subzero temperatures implies a switch to glycolysis. A threefold higher 13C recovery in microbial biomass at -5 than +5°C points to synthesis of intracellular compounds such as glycerol and ethanol in response to freezing. Less than 0.4% of 13C was recovered in DOC after 1 day, demonstrating complete glucose uptake by microorganisms even at -20°C. Consequently, we attribute the fivefold higher extracellular 13C in soil than in microbial biomass to secreted antifreeze compounds. This suggests that with decreasing temperature, intracellular antifreeze protection is complemented by extracellular mechanisms to avoid cellular damage by crystallizing water. The knowledge of sustained metabolism at subzero temperatures will not only be useful for modeling global C dynamics in ecosystems with periodically or permanently frozen soils, but will also be important in understanding and controlling the adaptive mechanisms of food spoilage organisms.

Soil microorganisms can overcome respiration inhibition by coupling intra- and extracellular metabolism: 13C metabolic tracing reveals the mechanisms

CO2 release from soil is commonly used to estimate toxicity of various substances on microorganisms. However, the mechanisms underlying persistent CO2 release from soil exposed to toxicants inhibiting microbial respiration, for example, sodium azide (NaN3) or heavy metals (Cd, Hg, Cu), remain unclear. To unravel these mechanisms, NaN3-amended soil was incubated with position-specifically 13C-labeled glucose and 13C was quantified in CO2, bulk soil, microbial biomass and phospholipid fatty acids (PLFAs). High 13C recovery from C-1 in CO2 indicates that glucose was predominantly metabolized via the pentose phosphate pathway irrespective of inhibition. Although NaN3 prevented 13C incorporation into PLFA and decreased total CO2 release, 13C in CO2 increased by 12% compared with control soils due to an increased use of glucose for energy production. The allocation of glucose-derived carbon towards extracellular compounds, demonstrated by a fivefold higher 13C recovery in bulk soil than in microbial biomass, suggests the synthesis of redox active substances for extracellular disposal of electrons to bypass inhibited electron transport chains within the cells. PLFA content doubled within 10 days of inhibition, demonstrating recovery of the microbial community. This growth was largely based on recycling of cost-intensive biomass compounds, for example, alkyl chains, from microbial necromass. The bypass of intracellular toxicity by extracellular electron transport permits the fast recovery of the microbial community. Such efficient strategies to overcome exposure to respiration-inhibiting toxicants may be exclusive to habitats containing redox-sensitive substances. Therefore, the toxic effects of respiration inhibitors on microorganisms are much less intensive in soils than in pure cultures.