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

Phlorizin released by apple root debris is related to apple replant disease

Autotoxic compounds are likely to be among the causes of apple replant disease, but their secretion is low during plant life. Using targeted metabolomics, the changes in soil phenolic profile were analyzed after the addition of apple roots, and their potential autotoxicity was assessed on apple seedlings. The addition of apple roots severely damaged the plants, attributed to autotoxic action of the phenolic compound phlorizin. Prolonged residence time of the roots in the soil before planting reduced their negative action, probably due to the degradation of phlorizin

Soil carbonyl sulfide exchange in relation to microbial community composition: Insights from a managed grassland soil amendment experiment

The viability of carbonyl sulfide (COS) measurements for partitioning ecosystem-scale net carbon dioxide (CO2) fluxes into photosynthesis and respiration critically depends on our knowledge of non-leaf sinks and sources of COS in ecosystems. We combined soil gas exchange measurements of COS and CO2 with next-generation sequencing technology (NGS) to investigate the role of soil microbiota for soil COS exchange. We applied different treatments (litter and glucose addition, enzyme inhibition and gamma sterilization) to soil samples from a temperate grassland to manipulate microbial composition and activity. While untreated soil was characterized by consistent COS uptake, other treatments reduced COS uptake and even turned the soil into a net COS source. Removing biotic processes through sterilization led to positive or zero fluxes. We used NGS to link changes in the COS response to alterations in the microbial community composition, with bacterial data having a higher explanatory power for the measured COS fluxes than fungal data. We found that the genera Arthrobacter and Streptomyces were particularly abundant in samples exhibiting high COS emissions. Our results indicate cooccurring abiotic production and biotic consumption of COS in untreated soil, the latter linked to carbonic anhydrase activity, and a strong dependency of the COS flux on the activity, identity, abundance of and substrate available to microorganisms.Austrian National Science Fund (FWF) | Ref. P27176-B16Tyrolean Science Fund (TWF) | Ref. UNI-0404/1801Ministerio de Economía 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

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

No time to die : comparative study on preservation protocols for anaerobic fungi

Anaerobic fungi (AF, phylum Neocallimastigomycota) are best known for their ability to anaerobically degrade recalcitrant lignocellulosic biomass through mechanic and enzymatic means. While their biotechnological potential is well-recognized, applied research on AF is still hampered by the time-consuming and cost-intensive laboratory routines required to isolate, maintain, and preserve AF cultures. Reliable long-term preservation of specific AF strains would aid basic as well as applied research, but commonly used laboratory protocols for AF preservation can show erratic survival rates and usually exhibit only moderate resuscitation success for up to one or two years after preservation. To address both, the variability, and the preservation issues, we have set up a cross-laboratory, year-long study. We tested five different protocols for the preservation of AF. The experiments were performed at three different laboratories (Austria, Germany, Switzerland) with the same three morphologically distinct AF isolates (Anaeromyces mucronatus, Caeocmyces sp., and Neocallimastix cameroonii) living in stable co-culture with their naturally occurring, syntrophic methanogens. We could show that handling greatly contributes to the variability of results, especially in Anaeromyces mucronatus. Cryopreservation of (mature) biomass in liquid nitrogen had the highest overall survival rates (85–100%, depending on the strain and laboratory). Additionally, preservation on agar at 39°C had surprisingly high survival rates for up to 9 months, if pieces of agar containing mature AF thalli were resuscitated. This low-cost, low-effort method could replace consecutive batch cultivation for periods of up to 6 months, while long-term preservation is best done by cryopreservation in liquid nitrogen. Regardless of the method, however, preserving several replicates (>three) of the same strain is highly advisable