The use of extracellular DNA as a proxy for specific microbial activity

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.publishersversionPeer reviewe

The potential of calcium hydroxide to reduce storage losses: A four months monitoring study of spruce wood chip piles at industrial scale

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 effect of calcium hydroxide on the storage behaviour of poplar wood chips in open-air piles

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 Güssing, 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

Establishment of a Laboratory Scale Set-Up with Controlled Temperature and High Humidity to Investigate Dry Matter Losses of Wood Chips from Poplar during Storage

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 setups 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

Characterization of organic-rich mineral debris revealed by rapid glacier retreat, Indren Glacier, European Alps

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

Soil slope exposure affects physico-chemical and microbiological properties in soil aggregate size fractions

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.Austrian Science Fund | Ref. I989-B16Ministerio de Economía, Industria y Competitividad | Ref. RYC-2016-2123

Soil slope exposure affects physico-chemical and microbiological properties in soil aggregate size fractions

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