Comparison of DNA extraction methods on different sample matrices within the same terrestrial ecosystem.

Metataxonomic studies of ecosystem microbiotas require the simultaneous processing of samples with contrasting physical and biochemical traits. However, there are no published studies of comparisons of different DNA extraction kits to characterize the microbiotas of the main components of terrestrial ecosystems. Here, and to our knowledge for the first time, five DNA extraction kits were used to investigate the composition and diversity of the microbiota of a subset of samples typically studied in terrestrial ecosystems such as bulk soil, rhizosphere soil, invertebrate taxa and mammalian feces. DNA extraction kit was associated with changes in the relative abundance of hundreds of ASVs, in the same samples, resulting in significant differences in alpha and beta diversity estimates of their microbiotas. Importantly, the impact of DNA extraction kit on sample diversity varies according to sample type, with mammalian feces and soil samples showing the most and least consistent diversity estimates across DNA extraction kits, respectively. We show that the MACHEREY-NAGEL NucleoSpin® Soil kit was associated with the highest alpha diversity estimates, providing the highest contribution to the overall sample diversity, as indicated by comparisons with computationally assembled reference communities, and is recommended to be used for any large-scale microbiota study of terrestrial ecosystems.

Primary study on frass fertilizers from mass-reared insects: Species variation, heat treatment effects, and implications for soil application at laboratory scale.

Insect farming has gained popularity as a resource-efficient and eco-friendly method for managing organic wastes by converting them into high-quality protein, fat, and frass. Insect frass is a powerful organic fertilizer that enriches the soil with essential plant nutrients and enhances plant defense mechanisms through chitin stimulation. Given the importance of frass commercialization for many insect farmers and the use of increasingly diverse organic wastes as insect feedstocks, there is a need for legal guidelines to enable clean production practices. The recent introduction of a legal definition for frass and heat treatment requirements by the EU commission marks a significant step towards standardizing its quality; however, little is known about the processes shaping its nutritional profiles and contributing to its maturation. Our study addresses key knowledge gaps in frass composition and production practices. Here, we analyzed the physicochemical, plant-nutritional, and microbiological properties of black soldier fly, yellow mealworm, and Jamaican field cricket frass from mass-rearing operations and assessed the impact of hygienizing heat treatment on fertilizer properties and frass microbiota. The results showed that frass properties varied significantly across insect species, revealing concentrations of plant-available nutrients as high as 7000 µg NH4+-N, 150 µg NO2-NO3--N, and 20 mg available P per g of total solids. Heat treatment reduced microbial activity, biomass, and viable counts of pathogenic Escherichia coli and Salmonella spp. In terms of frass microbiome composition, alpha diversity showed no significant differences between fresh and heat-treated frass samples; however, significant differences in microbial community composition were observed across the three insect species. Despite heat treatment, soil application of frass reactivated and boosted soil microbial activity, inducing up to a 25-fold increase in microbial respiration, suggesting no long-term detrimental effects on microorganisms. These findings not only enhance our understanding of insect frass as a nutrient-rich organic fertilizer but also have implications for regulatory frameworks, underscoring its promising potential for soil health and nutrient cycling. However, it is important to recognize the primary nature of this research, conducted at laboratory scale and over a short term. Future studies should aim to validate these findings in agricultural settings and explore additional factors influencing frass properties and its (long-term) interaction with soil ecosystems.

Microbial perspective of inhibited carbon turnover in Tangel humus of the Northern Limestone Alps.

Tangel humus primarily occurs in montane and subalpine zones of the calcareous Alps that exhibit low temperatures and high precipitation sums. This humus form is characterized by inhibited carbon turnover and accumulated organic matter, leading to the typical thick organic layers. However, the reason for this accumulation of organic matter is still unclear, and knowledge about the microbial community within Tangel humus is lacking. Therefore, we investigated the prokaryotic and fungal communities along with the physical and chemical properties within a depth gradient (0-10, 10-20, 20-30, 30-40, 40-50 cm) of a Tangel humus located in the Northern Limestone Alps. We hypothesized that humus properties and microbial activity, biomass, and diversity differ along the depth gradient and that microbial key players refer to certain humus depths. Our results give the first comprehensive information about microbiota within the Tangel humus and establish a microbial zonation of the humus. Microbial activity, biomass, as well as microbial alpha diversity significantly decreased with increasing depths. We identified microbial biomarkers for both, the top and the deepest depth, indicating different, microbial habitats. The microbial characterization together with the established nutrient deficiencies in the deeper depths might explain reduced C-turnover and Tangel humus formation.

Mock community as an in situ positive control for amplicon sequencing of microbiotas from the same ecosystem.

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Currently available protocols for metataxonomy assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. It has been suggested that the addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases during processing and support direct comparisons of microbiota composition, but the impact of MC on diversity estimates of samples is unknown. Here, large and small aliquots of pulverized bovine fecal samples were extracted with no, low or high doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We demonstrated that sample diversity estimates were distorted only if MC dose was high compared to sample mass (i.e. when MC > 10% of sample reads). We also showed that MC was an informative in situ positive control, permitting an estimation of the sample 16S copy number, and detecting sample outliers. We tested this approach on a range of sample types from a terrestrial ecosystem, including rhizosphere soil, whole invertebrates, and wild vertebrate fecal samples, and discuss possible clinical applications.

Soil function indicators are influenced by land use of different ages: A case study in a semi-arid region.

Soil function refers to all the roles and services that the soil performs, and can be assessed by measuring physical, chemical and biological properties. In any case, studies on the state of fertility and biological activities are now emphasized as indicators of soil functions. Degradation of rangelands and their conversion to agricultural land is one of the most common land use changes in different parts of the world, with significant impacts on soil functions. Not much is known about the effects of land use change (especially rangeland to orchards of different ages) on soil function indicators in semi-arid areas. In the current study, the following five types of land covers were considered in a semi-arid region of northern Iran: (1) enclosured rangeland, (2) 10-year-old apple orchard, (3) 25-year-old apple orchard, (4) 10-year-old walnut orchard, and (5) 25-year-old walnut orchard. Results showed that the apple orchards (25-year-old) had a more fertile soil and a higher biological activity than the other land uses studied. Based on heat plots of soil properties under different land uses, the apple orchards (25-year-old) formed hot spots of soil functional indicators in the study area, followed by the walnut orchard (25-year-old) > enclosured rangeland > apple orchard (10-year-old) > walnut orchard (10-year-old). Although the conversion of natural lands (i.e., rangelands) to anthropogenic ones (i.e., orchards) is generally associated with negative feedbacks, tree species can be used (by creating forested rangeland or a combination of rangeland-agriculture) in areas with low soil function to improve soil conditions (in the long term).

Carbon-dependent growth, community structure and methane oxidation performance of a soil-derived methanotrophic mixed culture.

Soil-borne methane-oxidizing microorganisms act as a terrestrial methane (CH4) sink and are potentially useful in decreasing global CH4 emissions. Understanding the ecophysiology of methanotrophs is crucial for a thorough description of global carbon cycling. Here, we report the in situ balance of soils from abandoned landfills, meadows and wetlands, their capacities to produce and oxidize CH4 at laboratory-scale and the isolation of a soil-borne methanotrophic-heterotrophic mixed culture that was used for carbon (C1 and C2) feeding experiments. We showed that even with similar soil properties, the in situ CH4 balance depends on land-use. Different soils had different potentials to adapt to increased CH4 availability, leading to the highest CH4 oxidation capacities for landfill and wetland soils. The most efficient mixed culture isolated from the landfill was dominated by the methanotrophs Methylobacter sp. and Methylosinus sp., which were accompanied by Variovorax sp. and Pseudomonas sp. and remained active in oxidizing CH4 when supplied with additional C-sources. The ratios between type I and type II methanotrophs and between methanotrophic and heterotrophic bacteria changed when C-sources were altered. A significant effect of the application of the mixed culture on the CH4 oxidation of soils was established but the extent varied depending on soil type.

Microbial community composition in the rhizosphere of Larix decidua under different light regimes with additional focus on methane cycling microorganisms.

Microbial community and diversity in the rhizosphere is strongly influenced by biotic and/or abiotic factors, like root exudates, nutrient availability, edaphon and climate. Here we report on the microbial diversity within the rhizosphere of Larix decidua, a dominant tree species in the Alps, as compared with the microbiome within the surrounding soil. We describe how increased light intensity influenced the rhizobiome and put emphasize on methane cycling microorganisms. Microbial taxa were classified into 26 bacterial, 4 archaeal and 6 fungal phyla revealing significant differences between bulk and rhizosphere soils. The dominant prokaryotic phyla were Proteobacteria, Acidobacteria, Actinobacteria (both, rhizosphere and bulk soil) and Bacteroidetes (rhizosphere soil only) and dominant fungal phyla in both fractions included Ascomycota and Basidiomycota. The rhizosphere community was indicated by Suillus sp., plant growth-promoting bacteria and Candidatus Saccharibacteria. Predicted genes in membrane transport and carbohydrate metabolism were significantly more abundant in rhizosphere soils while genes connected with energy metabolisms and cell motility increased in bulk soils. Dominant methanotrophic microorganisms were Upland Soil Cluster (USC) α methanotrophs, Methylogaea spp. and Methylosinus spp., while most methanogens belonged to Methanomassiliicoccales. The overall abundance of methanotrophs distinctly increased in the rhizosphere but to a very different species-specific extent. The increased light intensity only led to minor changes in the rhizobiome, nevertheless a couple of indicator species (e.g. Pseudomonas sp.) for intensified light conditions were established.

Soil-Derived Inocula Enhance Methane Production and Counteract Common Process Failures During Anaerobic Digestion.

Although soil-borne methanogens are known to be highly diverse and adapted to extreme environments, their application as potential (anaerobic) inocula to improve anaerobic digestion has not been investigated until now. The present study aimed at evaluating if soil-derived communities can be beneficial for biogas (methane, CH4) production and endure unfavorable conditions commonly associated with digestion failure. Nine study sites were chosen and tested for suitability as inoculation sources to improve biogas production via in situ measurements (CH4 fluxes, physical and chemical soil properties, and abundance of methanogens) and during a series of anaerobic digestions with (a) combinations of both sterile or unsterile soil and diluted fermenter sludge, and (b) pH-, acetate-, propionate-, and ammonium-induced disturbance. Amplicon sequencing was performed to assess key microbial communities pivotal for successful biogas production. Four out of nine tested soil inocula exerted sufficient methanogenic activity and repeatedly allowed satisfactory CH4/biogas production even under deteriorated conditions. Remarkably, the significantly highest CH4 production was observed using unsterile soil combined with sterile sludge, which coincided with both a higher relative abundance of methanogens and predicted genes involved in CH4 metabolism in these variants. Different bacterial and archaeal community patterns depending on the soil/sludge combinations and disturbance variations were established and these patterns significantly impacted CH4 production. Methanosarcina spp. seemed to play a key role in CH4 formation and prevailed even under stressed conditions. Overall, the results provided evidence that soil-borne methanogens can be effective in enhancing digestion performance and stability and, thus, harbor vast potential for further exploitation.

Spiking a Silty-Sand Reference Soil with Bacterial DNA: Limits and Pitfalls in the Discrimination of Live and Dead Cells When Applying Ethidium Monoazide (EMA) Treatment.

In the present study, EMA (ethidium monoazide) treatment was applied to a silty-sand reference soil prior to DNA extraction to enable a differentiation between dead and living cells. For this purpose, a reference soil was spiked with Listeria monocytogenes cells or cell equivalents, respectively. With the purpose of evaluating optimum treatment conditions, different EMA concentrations have been tested. However, the results remained largely inconclusive. Furthermore, varied dark incubation periods allowing EMA to penetrate dead cells did not allow the selective removal of DNA from membrane-compromised cells in downstream analyses. In contrast to undiluted soil, an effect of EMA treatment during DNA extraction could be observed when using a 1:10 dilution of the reference soil; however, the effect has not been sufficiently selective to act on heat-treated cells only. Although the application of EMA to soil requires further evaluation, the procedure harbors future potential for improving DNA-based approaches in microbial ecology studies.

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

Microbial Diversity in Bulk and Rhizosphere Soil of Ranunculus glacialis Along a High-Alpine Altitudinal Gradient.

Serving as "natural laboratories", altitudinal gradients can be used to study changes in the distribution of microorganisms in response to changing environmental conditions that typically occur over short geographical distances. Besides, rhizosphere zones of plants are known to be hot-spots for microbial diversity and to contain different microbial communities when compared with surrounding bulk soil. To discriminate the effects of altitude and plants, we investigated the microbial communities in the rhizosphere of Ranunculus glacialis and bulk soil along a high-alpine altitudinal gradient (2,600-3,400 m a.s.l.). The research area of this study was Mount (Mt.) "Schrankogel" in the Central Alps of Tyrol (Austria). Our results point to significantly different microbial diversities and community compositions in the different altitudinal belts. In the case of prokaryotes, environmental parameters could explain 41% of the total variation of soil communities, with pH and temperature being the strongest influencing factors. Comparing the effects derived from fraction (bulk vs. rhizosphere soil) and environmental factors, the effects of the roots of R. glacialis accounted for about one third of the explained variation. Fungal communities on the other hand were nearly exclusively influenced by environmental parameters accounting for 37.4% of the total variation. Both, for altitudinal zones as well as for bulk and rhizosphere fractions a couple of very specific biomarker taxa could be identified. Generally, the patterns of abundance of several taxa did not follow a steady increased or decreased trend along the altitudinal gradient but in many cases a maximal or minimal occurrence was established at mid-altitudes (3,000-3,100 m). This mid-altitudinal zone is a transition zone (the so-called alpine-nival ecotone) between the (lower) alpine grassland/tundra zone and the (upper) sparsely vegetated nival zone and was shown to correspond with the summer snow line. Climate change and the associated increase in temperature will shift this transition zone and thus, might also shift the described microbial patterns and biomarkers.

Larix decidua and additional light affect the methane balance of forest soil and the abundance of methanogenic and methanotrophic microorganisms.

Due to the activity of methane-oxidizing bacteria, forest soils are usually net sinks for the greenhouse gas methane (CH4). Despite several hints that CH4 balances might be influenced by vegetation, there are only few investigations dealing with this connection. Therefore, we studied this soil-plant-microbe interaction by using mesocosm experiments with forest soil and Larix decidua, a common coniferous tree species within the Alps. Gas measurements showed that the presence of L. decidua significantly reduced CH4 oxidation of the forest soil by ∼10% (-0.95 µmol m-2 h-1 for soil vs -0.85 µmol m-2 h-1 for soil plus L. decidua) leading to an increased net CH4 balance. Increased light intensity was used to intensify the influence of the plant on the soil's CH4 balance. The increase in light intensity strengthened the effect of the plant and led to a greater reduction of CH4 oxidation. Besides, we examined the impact of L. decidua and light on the abundance of methanogens and methanotrophs in the rhizosphere as compared with bulk soil. The abundance of both methane-oxidizing bacteria and methanogenic archaea was significantly increased in the rhizosphere compared with bulk soil but no significant response of methanogens and methanotrophs upon light exposure was established.

The influence of cattle grazing on methane fluxes and engaged microbial communities in alpine forest soils.

Recent dynamics and uncertainties in global methane budgets necessitate a dissemination of current knowledge on the controls of sources and sinks of atmospheric methane. Forest soils are considered to be efficient methane sinks; however, as they are microbially mediated they are sensitive to anthropogenic influences and tend to switch from being sinks to being methane sources. With regard to global changes in land use, the present study aimed at (i) investigating the influence of grazing on flux rates of methane in forest soils, (ii) deducing possible (a)biotic factors regulating these fluxes, and (iii) gaining an insight into the complex interactions between methane-cycling microorganisms and ecosystem functioning. Here we show that extensive grazing significantly mitigated the soil's sink strength for atmospheric methane through alterations of both microbial activity and community composition. In situ flux measurements revealed that all native, non-grazed areas were net methane consumers, while the adjacent, grazed areas were net methane producers. Whereas neither parent material nor soil properties including moisture and organic matter showed any correlation to the ascertained fluxes, significantly higher archaeal abundances at the grazed study sites indicated that small inputs of methanogens associated with cattle grazing may be sufficient to sustainably increase methane emissions.

Methane-cycling microorganisms in soils of a high-alpine altitudinal gradient.

Methanogens and methanotrophs play unique roles as producers and consumers of the greenhouse gas methane (CH4) in soils, respectively. Here, we aimed to reveal whether and to which extent methane-cyclers occur in high-alpine soils, and to assess their spatial distribution along an altitudinal gradient (2700-3500 m) in the Austrian Alps at sites located within the alpine (2700-2900 m), the alpine-nival (3000-3100 m) and the nival belts (3200-3500 m). Methanococcales and Methanocella spp. were most abundant among all quantified methanogenic guilds, whereas Methanosarcinales were not detected in the studied soil. The detected methanogens seem to be capable of persisting despite a highly oxic low-temperature environment. Methanogenic and methanotrophic activities and abundances of methanotrophs, Methanococcales and Methanocella spp. declined with altitude. Methanogenic and methanotrophic abundances were best explained by mean annual soil temperature and dissolved organic carbon, respectively. Alpine belt soils harbored significantly more methane-cyclers than those of the nival belt, indicating some influence of plant cover. Our results show that methanogens are capable of persisting in high-alpine cold soils and might help to understand future changes of these environments caused by climate warming.

Abundance and potential metabolic activity of methanogens in well-aerated forest and grassland soils of an alpine region.

Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities.

Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil.

Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol-chloroform-isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations.