The study of microbial communities based on the total environmental DNA (eDNA) is influenced by the presence of different eDNA states, i.e., intracellular (iDNA) and extracellular DNA (exDNA), and the choice of the DNA extraction method. Although the use of spike-and-recovery controls facilitates the diagnosis of such issues, appropriate experimental setups simultaneously accounting for the different eDNA states and their bacterial origins are missing. Here, we used two single-gene deletion mutants of both Escherichia coli and Bacillus subtilis to trace exDNA and iDNA spike-ins of each selected model organism within environmental samples. Unique primer/probe sets were developed for each strain, allowing their absolute quantification using multiplex digital PCR assays. The proposed spike-and-recovery controls were successfully applied to various environments including soil, sediment, sludge and compost. While the percent recovery of spiked iDNA differed significantly between E. coli and B. subtilis, results were similar for both model organisms in the case of spiked exDNA, emphasising that the fate of DNA molecules in the environment is similar irrespective of their bacterial origin. Hence, future studies may benefit from the proposed approach to better understand methodological ambiguities related to the eDNA extraction in general as well as the separation of the different eDNA states.

Soil microbes drive ecosystem function and play a critical role in how ecosystems respond to global change. Research surrounding soil microbial communities has rapidly increased in recent decades, and substantial data relating to phospholipid fatty acids (PLFAs) and potential enzyme activity have been collected and analysed. However, studies have mostly been restricted to local and regional scales, and their accuracy and usefulness are limited by the extent of accessible data. Here we aim to improve data availability by collating a global database of soil PLFA and potential enzyme activity measurements from 12,258 georeferenced samples located across all continents, 5.1% of which have not previously been published. The database contains data relating to 113 PLFAs and 26 enzyme activities, and includes metadata such as sampling date, sample depth, and soil pH, total carbon, and total nitrogen. This database will help researchers in conducting both global- and local-scale studies to better understand soil microbial biomass and function.

MikroMondo, a forthcoming science center in Austria, will be dedicated to the fascinating world of microorganisms, with a special focus on soil and its intricate biodiversity. Designed to promote soil literacy among the public, MikroMondo will feature cutting-edge exhibits, among them Europe's tallest Winogradsky column, live demonstrations of plant-microbe interactions (e.g., mycorrhization of pine seedlings in transparent soil columns), and engaging sound-producing slime molds. These exhibits aim to captivate visitors and highlight the vital role of microorganisms in soil ecosystems.The center will also offer interactive, hands-on activities tailored for school pupils, students, and educators, designed to deepen understanding of microbiological processes such as carbon and nutrient cycling, plant-microbe symbiosis, and soil microbial diversity. Activities will include guided microscopy sessions, microbial cultivation, the creation of Winogradsky columns, and decomposition and gas production experiments. MikroMondo’s innovative approach will inspire curiosity and enhance public appreciation of the hidden world beneath our feet, fostering a new generation of soil stewards and microbial enthusiasts.

The upcoming MicrobeX-Science Center in Zirl, close to Innsbruck (Tyrol, Austria), is focused on microorganisms governing our daily life. The storyline is crossing atmospheric microbiology related to climate change, food microbiology and environmental biotechnology like biomethanisation and wastewater treatment. A most central role, however, will have soil microbiology that is related to the effects of microorganisms on climate, in particular soil greenhouse gas production and uptake, plant-growth promoting rhizobacteria related to environmentally sound alternative agriculture, and also to microbially produced volatiles like geosmin, the scent of soil. Soil biodiversity will also be addressed, in relation to deadwood (until its microbial decomposition into the very first humus form, the so-called lignoform), soil-dwelling myxobacteria and erosion-preventing mycorrhizae. The talk will show how soil microorganisms will be embedded in a storyline that aims at promoting the public interest in microbiology, and microbes in geo- and in particular in soil sciences, with the challenging purpose of generally raising awareness about the central role of (soil) microbes in the past, presence and future.

Biomass degradation by microorganisms may cause major losses during the storage of wood chips for energy production. Poplar wood chips from short rotation coppices are especially prone to degradation with dry matter losses (DML) of up to 25% within a storage period, emphasizing the need for countermeasures. Therefore, we investigated the potential of the addition of alkaline Ca(OH)2 to the wet biomass of poplar wood chips and hypothesised that the establishment of an alkaline environment would reduce the activity of fungi, the primary wood degraders. Three industrial-scale piles (250 m3) with 0, 1.5 and 3% Ca(OH)2 were installed in Gussing, Austria and for four months (April-August 2019) the pile temperature, pH, moisture content, gas evolution (O2, CO2, H2, H2S, CH4) as well as DML were monitored. Ca(OH)2 altered the physicochemical properties of the wood chips but did not prevent biomass losses. However, as compared to literature, the DML were, compared to earlier investigations, also low in the control. In addition, cultivation methods were performed to evaluate the diversity of thermophilic microbes throughout the storage. Numerous filamentous fungi belonging to the phyla Ascomycota and Mucoromycota were isolated, being Rhizomucor pusillus, Aspergillus fumigatus, Thermomyces lanuginosa and Thermoascus aurantiacus the dominant species. Only minor differences in the fungal composition were detected as a result of Ca(OH)2 addition. Instead, clear shifts in colony forming units (CFUs) were detected as a function of progressing storage time, with a decrease of the number of propagules after four months.

The analysis of environmental DNA (eDNA) is revolutionizing the monitoring of biodiversity as it allows to assess organismic diversity at large scale and unprecedented taxonomic detail. However, eDNA consists of an extracellular and intracellular fraction, each characterized by particular properties that determine the retrievable information on when and where organisms live or have been living. Here, we review the fractions of eDNA, describe how to obtain them from environmental samples and present a four-scenario concept that aims at enhancing spatial and temporal resolution of eDNA-based monitoring. Importantly, we highlight how the appropriate choice of eDNA fractions precludes misinterpretation of eDNA-based biodiversity data. Finally, future avenues of research towards eDNA fraction-specific analyses are outlined to unravel the full potential of eDNA-based studies targeting micro- and macro-organisms.

Storage of woody biomass in large wood chip piles is unavoidable for biotechnological applications, but comes along with considerable biomass-, energy- and thus, economic losses due to exothermic reactions and microbial degradation. The homogeneous amendment of the storage piles with an alkaline stabilization agent, calcium hydroxide (Ca(OH)2), was found to decrease dry matter loss in Picea abies; for Populus canadensis piles the effects cannot clearly be deduced. Here we investigated the bacterial and fungal communities of industrial-scale wood chip piles (250 m3) of these two different tree species and related them to physicochemical conditions and enzymatic activities after 35 and 120 d, representing short- and long-term storage of the wood chips, respectively. Coming from different wood types (hard vs. softwood), we expected the communities to converge over time, due to similar storage conditions. Despite pH posing selective pressure, we expected a minor Ca(OH)2 effect as already known from previous studies. We found that the effectiveness of Ca(OH)2 addition depended on the wood type that determined both the native microbial seeding community and temperature pattern of all piles, thereby exerting selective forces of differing strength. Generally, a thermophilic community consisting of single fungal and variable bacterial taxa were identified. As expected, the microbial communities from P. abies and P. canadensis converged over time. Biomass loss was connected to C-cycle related enzymatic activities and to the abundance and composition of fungal communities. Chaetomium sp. was identified as potential key taxon determining biomass degradation under the given storage conditions.

Slope exposure is known to affect soil biogeochemical processes in mountainous forest ecosystems, but little attention has yet been paid to its influence at a soil aggregate scale. Therefore, we evaluated the effects of slope exposure (north- vs south-facing slope) on the physico-chemical and microbiological properties of bulk soil and dry-sieved and water-stable aggregate size fractions in both organic (OF) and mineral (AE) horizons in an Italian alpine forest. The changes in organic carbon (OC) and nitrogen (ON) fractions were assessed together with a battery of thirteen enzyme activities involved in the main nutrient cycles. In addition, soil biological properties including microbial biomass (estimated as double-stranded DNA content), and microbial activity (assessed as the ratio between the extra-(exDNA) and intracellular (iDNA) fractions of the total soil DNA pool) were determined. The OF horizon at the north-facing slope was enriched in recalcitrant and insoluble OC and ON fractions and characterized by a lower microbial activity, as indicated by the higher exDNA/iDNA ratio with respect to the south-facing slope. On the contrary, exDNA and iDNA contents, microbial biomass, as well as most of the enzyme activities, reached higher levels at the southern exposure in the AE horizon. These exposure-effects were bulk soil- and aggregate size fraction-specific. Overall, lower values of the chemical and microbiological parameters were found in the water-stable fraction. Our findings indicate that slope exposure (and thus topography), soil horizon, and aggregate size distinctly influence soil OC dynamics in mountain ecosystems.

The aim of this work was to improve the understanding of dry matter losses (DML) that occur in wood chips during the initial phase of storage in outdoor piles. For this purpose, a laboratory scale storage chamber was developed and investigated regarding its ability to recreate the conditions that chips undergo during the initial phase of outdoor storage. Three trials with poplar Max-4 (Populus maximowiczii Henry × Populus nigra L.) chips were performed for 6–10 weeks in the storage chamber under controlled temperature and assisted humidity. Two different set-ups were investigated to maintain a high relative humidity (RH) inside the storage chamber; one using water containers, and one assisted with a humidifier. Moisture content (MC) and DML of the chips were measured at different storage times to evaluate their storage behaviour in the chamber. Additionally, microbiological analyses of the culturable fraction of saproxylic microbiota were performed, with a focus on mesophilic fungi, but discriminating also xerophilic fungi, and mesophilic bacteria, with focus on actinobacteria, in two trials, to gain a view on the poplar wood chip-inhabiting microorganisms as a function of storage conditions (moisture, temperature) and time. Results show that DML up to 8.8–13.7% occurred in the chips within 6–10 storage weeks. The maximum DML were reached in the trial using the humidifier, which seemed a suitable technique to keep a high RH in the testing chamber, and thus, to analyse the wood chips in conditions comparable to those in outdoor piles during the initial storage phase.

Our question posed for and used as title of the special issue ‘Hide and Seek of Soil Microbes’–Who is Where with Whom and Why [...]

One of the most challenging aspects of using wood chips as renewable energy source is the loss of biomass related to storage. Therefore, we installed three outdoor industrial-scale piles (250 m(3)) of poplar wood chips and monitored the bacterial and fungal communities by next-generation sequencing over a storage period of 120 d. Two of the three piles were supplemented with calcium dihydroxide (Ca(OH)(2)) (1.5%, 3% w/w) in order to test its potential as alkaline stabilization agent to preserve woody biomass during storage. Shifts in the microbial community composition occurred almost entirely in the beginning of the storage experiment, which we attribute to the temperature rise of up to 60 degrees C within the first week of storage. Later, however, we found little changes. Independent of Ca(OH)(2) concentration, a consortium of lignocellulolytic and thermotolerant microorganisms dominated the stored wood chip microbiota emphasizing their role as key players during wood decomposition. Although the addition of Ca(OH)(2) altered the physicochemical properties of wood chips, it did not prevent loss of biomass. Especially the pH was increased in Ca(OH)(2) treated piles. However, only minor differences in the microbial communities' composition were detected following Ca(OH)(2) addition, highlighting the microbes tolerance towards and adaptation to changing environmental conditions.

Microbiome studies mostly rely on total DNA extracts obtained directly from environmental samples. The total DNA consists of both intra- and extracellular DNA, which differ in terms of their ecological interpretation. In the present study, we have investigated for the first time the differences among the three DNA types using microbiome sequencing of Picea abies deadwood logs (Hunter decay classes I, III, and V). While the bacterial compositions of all DNA types were comparable in terms of more abundant organisms and mainly depended on the decay class, we found substantial differences between DNA types with regard to less abundant amplicon sequence variants (ASVs). The analysis of the sequentially extracted intra- and extracellular DNA fraction, respectively, increased the ecological depth of analysis compared to the directly extracted total DNA pool. Both DNA fractions were comparable in proportions and the extracellular DNA appeared to persist in the P. abies deadwood logs, thereby causing its masking effect. Indeed, the extracellular DNA masked the compositional dynamics of intact cells in the total DNA pool. Our results provide evidence that the choice of DNA type for analysis might benefit a study’s answer to its respective ecological question. In the deadwood environment researched here, the differential analysis of the DNA types underlined the relevance of Burkholderiales , Rhizobiales and other taxa for P. abies deadwood decomposition and revealed that the role of Acidobacteriota under this scenario might be underestimated, especially compared to Actinobacteriota .

The combined action of physical protection and chemical stabilisation is the main factor regulating the persistence of soil organic matter (SOM). In particular, physical protection refers to the compartmentalization of SOM and microbial biomass within aggregates. Thus, we have investigated the role of (macro-, meso- and micro-) soil aggregates in the protection of SOM and extracellular and intracellular DNA (eDNA vs iDNA) from oxidative stress by treating aggregates with Low Temperature Ashing (LTA) for different durations (0, 5, 24 and 48 h). DNA (exDNA vs iDNA) was sequentially extracted from the different aggregate size classes, and qualitatively (agarose gel electrophoresis) and quantitatively (fluorimetry; spectrophotometry) analysed, discriminating between double stranded (ds) and single stranded (ss) DNA. In addition, comparative PCR-DGGE (exDNA vs iDNA) was performed to assess the bacterial, fungal and archaeal communities of the different aggregate size classes. The use of LTA enabled to determine the amount of physically protected DNA in different aggregate sizes, evidencing the potential involvement of both exDNA and iDNA in the formation of the aggregates. Our results also provided insights into the important role of soil aggregates in protecting the genetic information of the autochthonous soil microbiota. The PCoA of bacterial, archaeal and fungal DGGE patterns displayed contrasting results. While fungal and archaeal fingerprinting patterns revealed direct relationships with aggregate sizes and LTA treatment, the bacterial community was not affected by these factors. This suggests a selective action of the size of aggregates on the dominant soil microbial community members. Our findings indicate that aggregates provide a protective habitat for the soil microbial community against environmental stress conditions, such as oxidative stress. Further, the specific protection of the soil microbiota (bacteria vs archaea vs fungi) is directly correlated to the size distribution of the microbial community and the aggregate size classes.

In the summer of 2003 and 2004, characterized by a rapid glacier retreat, a stony surface covered by well-structured organic-rich mineral debris was observed very close to the Indren glacier terminus (Monte Rosa Massif, NW Italy, 3100 m ASL), on an area covered by the glacier tongue till the year before. The origin and type of this organic-rich material were investigated, in order to detect their characteristics, potential sources and fate within the foreland system. The deposits were dated using Carbon-14 and analyzed for the chemical characteristics of the organic component, the elemental composition of the mineral fraction and presence of microbial markers. The material, granular and dark in color, had a total organic carbon (TOC) content ranging between 17.4 ± 0.39 and 28.1 ± 0.63 g kg −1 dry weight (dw), significantly higher than the surrounding glacial till (~ 1.4 g kg −1 dw), although only 0.33% of it was in water soluble form. Microbial carbon (C) and nitrogen (N) accounted for 10.6% and 3.13% of TOC and total N, respectively. Dissolved nitrogen (N), mainly present as ammonium, represented 2.40% of the total N. The low aromatic component and large presence of nitrogen (N)-derived compounds suggested that most of the organic carbon (OC) in these organic-rich mineral deposits was derived from microbial cells, although the high average radiocarbon age of about 2900 years may also point to the contribution of aeolian depositions of anthropogenic or natural origin. Elemental composition and the crustal enrichment factor of trace elements in the mineral fraction of the aggregates corroborated the hypothesis that most part of the accumulated material derived from ice meltwater. Some indicators of the colonization of these deposits by microbial communities were also reported, from the abundance of DNA and phylogenetic markers, to the presence of bacterial taxa commonly able to thrive in similar habitats. All these elements suggested that such kind of deposits may have a potential role as energy and nutrient sources in recently deglaciated areas, highlighting the necessity to better understand the processes underlying their formation and their evolution.

The objective of this study was to investigate the effect of an alkaline additive on the storage of wood chips from Norway spruce forest residues. Piles of untreated and calcium hydroxide treated wood chips (250 m3) were set up and investigated for four months. It was demonstrated that adding Ca(OH)2 to moist wood chips decreased the dry matter loss by 6%. This was attributed to the increase of the pH to a level of 8, rendering the habitat less suitable for fungal colonisation. The results suggest the set-up storage strategy as a potential alternative method for preserving wood chips when long term storage is required.

The dwarf shrub Cressa cretica is a thermocosmopolitan halophilic species. Different mechanisms confer salt stress tolerance such as tissue and osmotic tolerance and ion exclusion, as well as the associated microbiota. The aims were (i) to investigate the best conditions for C. cretica seed germination and to examine the tolerance of germinated seeds and seedlings to different salt concentrations and (ii) to characterize the rhizosphere and bulk soil microbiota. Germination and growth experiments were conducted to address plant salt tolerance, and with Illumina sequencing the microbiota of rhizosphere and bulk soil was investigated. While high salt concentrations (600, 800, and 1000 mM NaCl) inhibited C.cretica seed germination, recovery of ungerminated seeds under non-saline conditions was high, indicating osmotic rather than toxic effects of high salt concentrations. The microbiota found in rhizosphere and bulk soil showed high similarity with that found in previous studies on halophyte-associated microbiota, among the Planctomyces, Halomonas and Jeotgalibacillus. Concluding, salt stress responses on the plant level were shown, as has the involvement of associated halotolerant bacteria. Still, the potential role for mitigating saline stress by the bacterial associates of C. cretica, most of them microbial dark matter, will have to be investigated, as will the contributions of archaea and fungi.