The masking effect of extracellular DNA and robustness of intracellular DNA in anaerobic digester NGS studies: A discriminatory study of the total DNA pool.

Most commonly, next generation sequencing-based microbiome studies are performed on the total DNA (totDNA) pool; however, this consists of extracellular- (exDNA) and intracellular (iDNA) DNA fractions. By investigating the microbiomes of different anaerobic digesters over time, we found that totDNA suggested lower species richness considering all and/or only common species and yielded fewer unique reads as compared to iDNA. Additionally, exDNA-derived sequences were more similar to those from totDNA than from iDNA and, finally, iDNA showed the best performance in tracking temporal changes in microbial communities. We postulate that abundant sequences present within the exDNA fraction mask the overall results of totDNA and provide evidence that exDNA has the potential to qualitatively bias microbiome studies at least in the anaerobic digester environment as it contains information about cells that were lysed hours or days ago. iDNA, however, was found to be more appropriate in providing reliable genetic information about potentially alive as well as rare microbes within the target habitat.

Bacterial communities of decaying Norway spruce follow distinct slope exposure and time-dependent trajectories.

Deadwood decay employs a complex metabolism and provides carbon and nutrients for soils. Although being highly diverse, the contribution of the bacterial deadwood colonizing community is underexplored compared with the fungal one. Therefore, we performed an in-field mesocosm study and monitored the bacterial communities in decaying experimental Picea abies wood blocks and their underlying soil on north- and south- exposed slopes in the Italian Alps over a 2-year period. The faster deadwood decay at the south-facing slope was associated with a higher bacterial richness and a higher number of specialist operational taxonomic units (OTUs) which were more strongly correlated to environmental parameters than other bacterial community members. With progressing decay, the wood and soil bacterial communities became more similar in terms of richness, diversity and evenness and especially at the south-facing slope, they also became more similar in terms of community composition. Exposure-specific OTUs suggest wood-soil interaction. However, despite the strong influence of exposure on the soil bacterial communities, the P. abies wood blocks shared a comparably high number of OTUs with the soil irrespective of the slope. At finer taxonomic scale, we identified Pseudomonas, Microbacteria, Sphingomonas, Xanthomonas, Methylovirgula and Burkholderia as decay associated, although their functional role needs further studies.

Extracellular DNA in natural environments: features, relevance and applications.

Extracellular DNA (exDNA) is abundant in many habitats, including soil, sediments, oceans and freshwater as well as the intercellular milieu of metazoa. For a long time, its origin has been assumed to be mainly lysed cells. Nowadays, research is collecting evidence that exDNA is often secreted actively and is used to perform a number of tasks, thereby offering an attractive target or tool for biotechnological, medical, environmental and general microbiological applications. The present review gives an overview on the main research areas dealing with exDNA, depicts its inherent origins and functions and deduces the potential of existing and emerging exDNA-based applications. Furthermore, it provides an overview on existing extraction methods and indicates common pitfalls that should be avoided whilst working with exDNA.

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.

Why eDNA fractions need consideration in biomonitoring.

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.

The Molecular Information About Deadwood Bacteriomes Partly Depends on the Targeted Environmental DNA.

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.

Quantities of Intra- and Extracellular DNA Reveal Information About Activity and Physiological State of Methanogenic Archaea.

Although being a common aim of many microbial ecology studies, measuring individual physiological conditions of a microbial group or species within a complex consortium is still a challenge. Here, we propose a novel approach that is based on the quantification of sequentially extracted extracellular (exDNA) and intracellular DNA (iDNA) and reveals information about cell lysis and activity of methanogenic archaea within a biogas-producing microbial community. We monitored the methane production rates of differently treated batch anaerobic cultures and compared the concentrations of the alpha subunit of the methyl coenzyme M reductase gene of methanogenic archaea in extracellular and intracellular DNA fractions and in the classically extracted total DNA pool. Our results showed that this fine-tuned DNA approach coupled with the interpretation of the ratio between free exDNA and iDNA considerably improved microbial activity tracking compared to the classical extraction/quantification of total DNA. Additionally, it allowed to identify and quantify methanogenic populations that are inactive and those that are strongly influenced by cell lysis. We argue that despite the need of further studies, this method represents a novel approach to gain specific physiological information from a complex environmental sample and holds the potential to be applied to other microbes of interest.

Preliminary evidences of the presence of extracellular DNA single stranded forms in soil.

The relevance of extracellular DNA (eDNA) in the soil ecosystem is becoming more and more evident to the scientific community by the progressive discovery of functions accompanying to natural gene transformation. However, despite the increased number of published articles dedicated to eDNA in soil, so far only few are focused on its single stranded form (eDNAss). The present paper is the first to investigate the quantitative relevance of eDNAss in the total soil eDNA pool, discriminating between its linear (eDNAssl) and circular (eDNAssc) forms and the respective weakly (wa) and tightly (ta) adsorbed fractions. The results showed the prevalence of eDNAss and its linear form in both the total soil eDNA pool and its wa and ta fractions. Both of the eDNAss fractions (linear and circular) were characterized by small fragments.

Publisher Correction: Fungal communities and their association with nitrogen-fixing bacteria affect early decomposition of Norway spruce deadwood.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fungal communities and their association with nitrogen-fixing bacteria affect early decomposition of Norway spruce deadwood.

Deadwood decomposition is relevant in nature and wood inhabiting fungi (WIF) are its main decomposers. However, climate influence on WIF community and their interactions with bacteria are poorly understood. Therefore, we set up an in-field mesocosm experiment in the Italian Alps and monitored the effect of slope exposure (north- vs. south-facing slope) on the decomposition of Picea abies wood blocks and their microbiome over two years. Unlike fungal richness and diversity, we observed compositional and functional differences in the WIF communities as a function of exposure. Wood-degrading operational taxonomic units (OTUs) such as Mycena, and mycorrhizal and endophytic OTUs were characteristic of the south-facing slope. On the north-facing one, Mucoromycota, primarily Mucor, were abundant and mixotrophic basidiomycetes with limited lignin-degrading capacities had a higher prevalence compared to the southern slope. The colder, more humid conditions and prolonged snow-coverage at north exposure likely influenced the development of the wood-degrading microbial communities. Networks between WIF and N2-fixing bacteria were composed of higher numbers of interacting microbial units and showed denser connections at the south-facing slope. The association of WIF to N2-fixing Burkholderiales and Rhizobiales could have provided additional competitive advantages, especially for early wood colonization.

Effects of slope exposure on soil physico-chemical and microbiological properties along an altitudinal climosequence in the Italian Alps.

Due to their sensitivity to changing environmental conditions sub- and alpine soils are often monitored in the context of climate change, usually, however, neglecting slope exposure. Therefore, we set up a climosequence-approach to study the effect of exposure and, in general, climate, on the microbial biomass and microbial diversity and activity, comprising five pairs of north (N)- and south (S)-facing sites along an altitudinal gradient ranging from 1200 to 2400m a.s.l. in the Italian Alps (Trentino Alto Adige, Italy). Soil physico-chemical properties were related to microbiological properties (microbial biomass: double strand DNA yield vs. substrate-induced respiration; diversity of bacterial, fungal and archaeal communities: genetic fingerprinting DGGE vs. real-time PCR; microbial activity: basal respiration vs. multiple hydrolytic enzyme assays) to monitor shifts in the diversity and activity of microbial communities as a function of slope exposure and to evaluate the most determinant chemical parameters shaping the soil microbiota. The exposure-effect on several hydrolytic key-enzymes was enzyme-specific: e.g. acid phosphomonoesterase potential activity was more pronounced at the N-facing slope while the activities of alkaline phosphomonoesterase, pyrophosphate-phosphodiesterase and arylsulfatase were higher at the S-facing slope. Furthermore, this exposure-effect was domain-specific: bacteria (S>N, altitude-independent); fungi (N~S); and archaea (N>S; altitude-dependent). Additionally, the abiotic parameters shaping the community composition were in general depending on soil depth. Our multidisciplinary approach allowed us to survey the exposure and altitudinal effects on soil physico-chemical and microbiological properties and thus unravel the complex multiple edaphic factor-effects on soil microbiota in mountain ecosystems.

Soil attributes and microclimate are important drivers of initial deadwood decay in sub-alpine Norway spruce forests.

Deadwood is known to significantly contribute to global terrestrial carbon stocks and carbon cycling, but its decay dynamics are still not thoroughly understood. Although the chemistry of deadwood has been studied as a function of decay stage in temperate to subalpine environments, it has generally not been related to time. We therefore studied the decay (mass of deadwood, cellulose and lignin) of equal-sized blocks of Picea abies wood in soil-mesocosms over two years in the Italian Alps. The 8 sites selected were along an altitudinal sequence, reflecting different climate zones. In addition, the effect of exposure (north- and south-facing slopes) was taken into account. The decay dynamics of the mass of deadwood, cellulose and lignin were related to soil parameters (pH, soil texture, moisture, temperature) and climatic data. The decay rate constants of Picea abies deadwood were low (on average between 0.039 and 0.040y(-1)) and of lignin close to zero (or not detectable), while cellulose reacted much faster with average decay rate constants between 0.110 and 0.117y(-1). Our field experiments showed that local scale factors, such as soil parameters and topographic properties, influenced the decay process: higher soil moisture and clay content along with a lower pH seemed to accelerate wood decay. Interestingly, air temperature negatively correlated with decay rates or positively with the amount of wood components on south-facing sites. It exerted its influence rather on moisture availability, i.e. the lower the temperature the higher the moisture availability. Topographic features were also relevant with generally slower decay processes on south-facing sites than on north-facing sites owing to the drier conditions, the higher pH and the lower weathering state of the soils (less clay minerals). This study highlights the importance of a multifactorial consideration of edaphic parameters to unravel the complex dynamics of initial wood decay.