Coupling of metataxonomics and culturing improves bacterial diversity characterization and identifies a novel Rhizorhapis sp. with metal resistance potential in a multi-contaminated waste sediment.

Long-term contaminated environments have been recognized as potential hotspots for bacterial discovery in taxonomic and functional terms for bioremediation purposes. Here, bacterial diversity in waste sediment collected from a former industrial dumpsite and contaminated with petroleum hydrocarbon and heavy metals was investigated through the parallel application of culture-independent (16S rRNA gene amplicon sequencing) and -dependent (plate culturing followed by colony picking and identification of isolates by 16S rRNA gene Sanger sequencing) approaches. The bacterial diversities retrieved by both approaches greatly differed. Bacteroidetes and Proteobacteria were dominant in the culture-independent community, while Firmicutes and Actinobacteria were the main culturable groups. Only 2.7% of OTUs (operational taxonomic units) in the culture-independent dataset were cultured. Most of the culturable OTUs were absent or in very low abundances in the culture-independent dataset, revealing that culturing is a useful tool to study the rare bacterial biosphere. One culturable OTUs (comprising only the isolate SPR117) was identified as a potential new species in the genus Rhizorhapis (class Alphaproteobacteria) and was selected for further characterization. Phytopathogenicity tests showed that Rhizorhapis sp. strain SPR117 (ATCC TSD-228) is not pathogenic to lettuce, despite the only described species in this genus, Rhizorhapis suberifaciens, is causal agent of the lettuce corky root disease. The genome of the strain SPR117 was sequenced, assembled in 256 contigs, with a length of 4,419,522 bp and a GC content of 59.9%, and its further annotation revealed the presence of genes related to the resistance to arsenic, copper, iron, and mercury, among other metals. Therefore, the coupling of metataxonomics and culturing is a useful tool to obtain not only an improved description of bacterial communities in contaminated environments, but also to isolate microorganisms with bioremediation potential.

Application of dry olive residue-based biochar in combination with arbuscular mycorrhizal fungi enhances the microbial status of metal contaminated soils.

Biochar made-up of dry olive residue (DOR), a biomass resulting from the olive oil extraction industry, has been proposed to be used as a reclamation agent for the recovery of metal contaminated soils. The aim of the present study was to investigate whether the soil application of DOR-based biochar alone or in combination with arbuscular mycorrhizal fungi (AMF) leads to an enhancement in the functionality and abundance of microbial communities inhabiting metal contaminated soils. To study that, a greenhouse microcosm experiment was carried out, where the effect of the factors (i) soil application of DOR-based biochar, (ii) biochar pyrolysis temperature (considering the variants 350 and 500 °C), (iii) soil application dose of biochar (2 and 5%), (iv) soil contamination level (slightly, moderately and highly polluted), (v) soil treatment time (30, 60 and 90 days) and (vi) soil inoculation with Funneliformis mosseae (AM fungus) on ß-glucosidase and dehydrogenase activities, FA (fatty acid)-based abundance of soil microbial communities, soil glomalin content and AMF root colonization rates of the wheat plants growing in each microcosm were evaluated. Biochar soil amendment did not stimulate enzyme activities but increased microbial abundances. Dehydrogenase activity and microbial abundances were found to be higher in less contaminated soils and at shorter treatment times. Biochar pyrolysis temperature and application dose differently affected enzyme activities, but while the first factor did not have a significant effect on glucosidase and dehydrogenase, a higher biochar dose resulted in boosted microbial abundances. Soil inoculation with F. mosseae favored the proliferation of soil AMF community and increased soil glomalin content as well as rates of AMF root colonization. This factor also interacted with many of the others evaluated to significantly affect soil enzyme activities, microbial abundances and AMF community. Our results indicate that the application of DOR-based biochar along with AMF fungi is an appropriate approach to improve the status of microbial communities in soils with a moderate metal contamination at short-term.

Priming effects in soils across Europe.

Land use is a key factor driving changes in soil carbon (C) cycle and contents worldwide. The priming effect (PE)-CO2 emissions from changed soil organic matter decomposition in response to fresh C inputs-is one of the most unpredictable phenomena associated with C cycling and related nutrient mobilization. Yet, we know very little about the influence of land use on soil PE across contrasting environments. Here, we conducted a continental-scale study to (i) determine the PE induced by 13 C-glucose additions to 126 cropland and seminatural (forests and grasslands) soils from 22 European countries; (ii) compare PE magnitude in soils under various crop types (i.e., cereals, nonpermanent industrial crops, and orchards); and (iii) model the environmental factors influencing PE. On average, PEs were negative in seminatural (with values ranging between -60 and 26 µg C g-1 soil after 35 days of incubation; median = -11) and cropland (from -55 to 27 µC g-1 soil; median = -4.3) soils, meaning that microbial communities preferentially switched from soil organic C decomposition to glucose mineralization. PE was significantly less negative in croplands compared with seminatural ecosystems and not influenced by the crop type. PE was driven by soil basal respiration (reflecting microbial activity), microbial biomass C, and soil organic C, which were all higher in seminatural ecosystems compared with croplands. This cross European experimental and modeling study elucidated that PE intensity is dependent on land use and allowed to clarify the factors regulating this important C cycling process.

The effects of ozone treatments on the agro-physiological parameters of tomato plants and the soil microbial community.

Ozone has been applied in many processes (drinking water disinfection and wastewater treatment, among others) based on its high degree of effectiveness as a wide-spectrum disinfectant and its potential for the degradation of pollutants and pesticides. Nevertheless, the effects of irrigation with ozonated water on the soil microbial community and plant physiology and productivity at the field scale are largely unknown. Here, we assessed the impact of irrigation with ozonated water on the microbial community of a Mediterranean soil and on Solanum lycopersicum L. agro-physiology and productivity in a greenhouse experiment. For this purpose, we evaluated: i) soil physicochemical properties, soil enzyme activities, and the biomass (through analysis of microbial fatty acids) and diversity (through 16S rRNA gene and ITS2 amplicon sequencing) of the soil microbial community, and ii) the nutrient content, physiology, yield, and fruit quality of tomato plants. Overall, the soil physicochemical properties were slightly affected by the treatments applied, showing some differences between continuous and intermittent irrigation with ozonated water. Only the soil pH was significantly reduced by continuous irrigation with ozonated water at the end of the assay. Biochemical parameters (enzymatic activities) showed no significant differences between the treatments studied. The biomasses of Gram- bacteria and fungi were decreased by intermittent and continuous irrigation with ozonated water, respectively. However, the diversity, structure, and composition of the soil microbial community were not affected by the ozone treatments. Changes in soil properties slightly affected tomato plant physiology but did not affect yield or fruit quality. The stomatal conductance was reduced and the intrinsic water use efficiency was increased by continuous irrigation with ozonated water. Our results suggest that soil health and fertility were not compromised, however ozonated water treatments should be tailored to individual crop conditions to avoid adverse effects.

Special Issue "Microorganisms in Recycling and Valorization of Organic Waste for Sustainable Soil Health and Management".

Anthropogenic activity generates huge amounts of solid organic wastes [...].

Studying Microbial Communities through Co-Occurrence Network Analyses during Processes of Waste Treatment and in Organically Amended Soils: A Review.

Organic wastes have the potential to be used as soil organic amendments after undergoing a process of stabilization such as composting or as a resource of renewable energy by anaerobic digestion (AD). Both composting and AD are well-known, eco-friendly approaches to eliminate and recycle massive amounts of wastes. Likewise, the application of compost amendments and digestate (the by-product resulting from AD) has been proposed as an effective way of improving soil fertility. The study of microbial communities involved in these waste treatment processes, as well as in organically amended soils, is key in promoting waste resource efficiency and deciphering the features that characterize microbial communities under improved soil fertility conditions. To move beyond the classical analyses of metataxonomic data, the application of co-occurrence network approaches has shown to be useful to gain insights into the interactions among the members of a microbial community, to identify its keystone members and modelling the environmental factors that drive microbial network patterns. Here, we provide an overview of essential concepts for the interpretation and construction of co-occurrence networks and review the features of microbial co-occurrence networks during the processes of composting and AD and following the application of the respective end products (compost and digestate) into soil.

The structure and function of soil archaea across biomes.

We lack a predictive understanding of the environmental drivers determining the structure and function of archaeal communities as well as the proteome associated with these important soil organisms. Here, we characterized the structure (by 16S rRNA gene sequencing) and function (by metaproteomics) of archaea from 32 soil samples across terrestrial ecosystems with contrasting climate and vegetation types. Our multi-"omics" approach unveiled that genes from Nitrosophaerales and Thermoplasmata dominated soils collected from four continents, and that archaea comprise 2.3 ± 0.3% of microbial proteins in these soils. Aridity positively correlated with the proportion of Nitrosophaerales genes and the number of archaeal proteins. The interaction of climate x vegetation shaped the functional profile of the archaeal community. Our study provides novel insights into the structure and function of soil archaea across climates, and highlights that these communities may be influenced by increasing global aridity.

Methane production by anaerobic co-digestion of mixed agricultural waste: cabbage and cauliflower.

Anaerobic co-digestion of residual cabbage and cauliflower mixed at a ratio 1:1 (w/w) was investigated in two continuously stirred tank reactors under mesophilic conditions to ensure stability and enhanced methane generation. The experiments, including start-up, inoculum acclimatisation and treatment of the waste mixture, were carried out over a 65-day period. The characterisation results showed that the residual mixture contained a high proportion of total Kjeldahl nitrogen (around 37 g N/kg dry weight). The maximum value of methanogenic yield potential was found to be 250 LSTP/kg VS (volatile solid) added, at STP conditions (0°C, 1 atm), by loading organic substrate at a concentration of 1 g VS/L, while its biodegradability was 60%. However, instability of the biomethanisation process was observed after 17 days, which might be a consequence of the high concentration of nitrogen in the reactors. The evaluation of the kinetics of the valorisation process revealed that the waste mixture studied can easily be biodegraded through anaerobic co-digestion.

Foliar mycoendophytome of an endemic plant of the Mediterranean biome (Myrtus communis) reveals the dominance of basidiomycete woody saprotrophs.

The true myrtle, Myrtus communis, is a small perennial evergreen tree that occurs in Europe, Africa, and Asia with a circum-Mediterranean geographic distribution. Unfortunately, the Mediterranean Forests, where M. communis occurs, are critically endangered and are currently restricted to small fragmented areas in protected conservation units. In the present work, we performed, for the first time, a metabarcoding study on the spatial variation of fungal community structure in the foliar endophytome of this endemic plant of the Mediterranean biome, using bipartite network analysis as a model. The local bipartite network of Myrtus communis individuals and their foliar endophytic fungi is very low connected, with low nestedness, and moderately high specialization and modularity. Similar network patterns were also retrieved in both culture-dependent and amplicon metagenomics of foliar endophytes in distinct arboreal hosts in varied biomes. Furthermore, the majority of putative fungal endophytes species were basidiomycete woody saprotrophs of the orders Polyporales, Agaricales, and Hymenochaetales. Altogether, these findings suggest a possible adaptation of these wood-decaying fungi to cope with moisture limitation and spatial scarcity of their primary substrate (dead wood), which are totally consistent with the predictions of the viaphytism hypothesis that wood-decomposing fungi inhabit the internal leaf tissue of forest trees in order to enhance dispersal to substrates on the forest floor, by using leaves as vectors and as refugia, during periods of environmental stress.

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.

Enhancing laccase production by white-rot fungus Funalia floccosa LPSC 232 in co-culture with Penicillium commune GHAIE86.

To obtain enzymatic preparations with higher laccase activity levels from Funalia floccosa LPSC 232, available for use in several applications, co-cultures with six filamentous microfungi were tested. A laccase non-producing soil fungus, identified as Penicillium commune GHAIE86, showed an outstanding ability to increase laccase activity (3-fold as compared to that for monoculture) when inoculated in 6-day-old F. floccosa cultures. Maximum laccase production with the F. floccosa and P. commune co-culture reached 60 U/mL, or twice that induced by chemical treatments alone. Our study demonstrated that co-culture with soil fungi might be a promising method for improving laccase production in F. floccosa. Although the enhancement of laccase activity was a function of P. commune inoculation time, two laccase isoenzymes produced by F. floccosa remained unchanged when strains were co-cultured. These data are compatible with the potential of F. floccosa in agricultural applications in soil, whose enzyme machinery could be activated by soil fungi such as P. commune.

Insights into microbial communities mediating the bioremediation of hydrocarbon-contaminated soil from an Alpine former military site.

The study of microbial communities involved in soil bioremediation is important to identify the specific microbial characteristics that determine improved decontamination rates. Here, we characterized bacterial, archaeal, and fungal communities in terms of (i) abundance (using quantitative PCR) and (ii) taxonomic diversity and structure (using Illumina amplicon sequencing) during the bioremediation of long-term hydrocarbon-contaminated soil from an Alpine former military site during 15 weeks comparing biostimulation (inorganic NPK fertilization) vs. natural attenuation and considering the effect of temperature (10 vs. 20 °C). Although a considerable amount of total petroleum hydrocarbon (TPH) loss could be attributed to natural attenuation, significantly higher TPH removal rates were obtained with NPK fertilization and at increased temperature, which were related to the stimulation of the activities of indigenous soil microorganisms. Changing structures of bacterial and fungal communities significantly explained shifts in TPH contents in both natural attenuation and biostimulation treatments at 10 and 20 °C. However, archaeal communities, in general, and changing abundances and diversities in bacterial and fungal communities did not play a decisive role on the effectiveness of soil bioremediation. Gammaproteobacteria and Bacteroidia classes, within bacterial community, and undescribed/novel groups, within fungal community, proved to be actively involved in TPH removal in natural attenuation and biostimulation at both temperatures.

Characterization of soil bacterial, archaeal and fungal communities inhabiting archaeological human-impacted layers at Monte Iato settlement (Sicily, Italy).

Microbial communities in human-impacted soils of ancient settlements have been proposed to be used as ecofacts (bioindicators) of different ancient anthropogenic activities. In this study, bacterial, archaeal and fungal communities inhabiting soil of three archaic layers, excavated at the archaeological site on Monte Iato (Sicily, Italy) and believed to have been created in a chronological order in archaic times in the context of periodic cultic feasts, were investigated in terms of (i) abundance (phospholipid fatty acid (PLFA) analysis and quantitative PCR)), (ii) carbon(C)-source consumption patterns (Biolog-Ecoplates) and (iii) diversity and community composition (Illumina amplicon sequencing). PLFA analyses demonstrated the existence of living bacteria and fungi in the soil samples of all three layers. The upper layer showed increased levels of organic C, which were not concomitant with an increment in the microbial abundance. In taxonomic terms, the results indicated that bacterial, archaeal and fungal communities were highly diverse, although differences in richness or diversity among the three layers were not detected for any of the communities. However, significantly different microbial C-source utilization patterns and structures of bacterial, archaeal and fungal communities in the three layers confirmed that changing features of soil microbial communities reflect different past human activities.

Seasonal soil microbial responses are limited to changes in functionality at two Alpine forest sites differing in altitude and vegetation.

The study of soil microbial responses to environmental changes is useful to improve simulation models and mitigation strategies for climate change. We here investigated two Alpine forest sites (deciduous forest vs. coniferous forest) situated at different altitudes (altitudinal effect) in spring and autumn (seasonal effect) regarding: (i) bacterial and fungal abundances (qPCR); (ii) diversity and structure of bacterial and fungal communities (amplicon sequencing); and (iii) diversity and composition of microbial functional gene community (Geochip 5.0). Significant altitudinal changes were detected in microbial abundances as well as in diversity and composition of taxonomic and functional communities as a consequence of the differences in pH, soil organic matter (SOM) and nutrient contents and soil temperatures measured between both sites. A network analysis revealed that deciduous forest site (at lower altitude) presented a lower resistance to environmental changes than that of coniferous forest site (at higher altitude). Significant seasonal effects were detected only for the diversity (higher values in autumn) and composition of microbial functional gene community, which was related to the non-significant increased SOM and nutrient contents detected in autumn respect to spring and the presumable high capacity of soil microbial communities to respond in functional terms to discreet environmental changes.

Near-Infrared Spectroscopy and Geostatistical Analysis for Modeling Spatial Distribution of Analytical Constituents in Bulk Animal By-Product Protein Meals.

Control and inspection operations within the context of safety and quality assessment of bulk foods and feeds are not only of particular importance, they are also demanding challenges, given the complexity of food/feed production systems and the variability of product properties. Existing methodologies have a variety of limitations, such as high costs of implementation per sample or shortcomings in early detection of potential threats for human/animal health or quality deviations. Therefore, new proposals are required for the analysis of raw materials in situ in a more efficient and cost-effective manner. For this purpose, a pilot laboratory study was performed on a set of bulk lots of animal by-product protein meals to introduce and test an approach based on near-infrared (NIR) spectroscopy and geostatistical analysis. Spectral data, provided by a fiber optic probe connected to a Fourier transform (FT) NIR spectrometer, were used to predict moisture and crude protein content at each sampling point. Variographic analysis was carried out for spatial structure characterization, while ordinary Kriging achieved continuous maps for those parameters. The results indicated that the methodology could be a first approximation to an approach that, properly complemented with the Theory of Sampling and supported by experimental validation in real-life conditions, would enhance efficiency and the decision-making process regarding safety and adulteration issues.

Microbiology Meets Archaeology: Soil Microbial Communities Reveal Different Human Activities at Archaic Monte Iato (Sixth Century BC).

Microbial ecology has been recognized as useful in archaeological studies. At Archaic Monte Iato in Western Sicily, a native (indigenous) building was discovered. The objective of this study was the first examination of soil microbial communities related to this building. Soil samples were collected from archaeological layers at a ritual deposit (food waste disposal) in the main room and above the fireplace in the annex. Microbial soil characterization included abundance (cellular phospholipid fatty acids (PLFA), viable bacterial counts), activity (physiological profiles, enzyme activities of viable bacteria), diversity, and community structure (bacterial and fungal Illumina amplicon sequencing, identification of viable bacteria). PLFA-derived microbial abundance was lower in soils from the fireplace than in soils from the deposit; the opposite was observed with culturable bacteria. Microbial communities in soils from the fireplace had a higher ability to metabolize carboxylic and acetic acids, while those in soils from the deposit metabolized preferentially carbohydrates. The lower deposit layer was characterized by higher total microbial and bacterial abundance and bacterial richness and by a different carbohydrate metabolization profile compared to the upper deposit layer. Microbial community structures in the fireplace were similar and could be distinguished from those in the two deposit layers, which had different microbial communities. Our data confirmed our hypothesis that human consumption habits left traces on microbiota in the archaeological evidence; therefore, microbiological residues as part of the so-called ecofacts are, like artifacts, key indicators of consumer behavior in the past.

Abundance and Diversity of Bacterial, Archaeal, and Fungal Communities Along an Altitudinal Gradient in Alpine Forest Soils: What Are the Driving Factors?

Shifts in soil microbial communities over altitudinal gradients and the driving factors are poorly studied. Their elucidation is indispensable to gain a comprehensive understanding of the response of ecosystems to global climate change. Here, we investigated soil archaeal, bacterial, and fungal communities at four Alpine forest sites representing a climosequence, over an altitudinal gradient from 545 to 2000 m above sea level (asl), regarding abundance and diversity by using qPCR and Illumina sequencing, respectively. Archaeal community was dominated by Thaumarchaeota, and no significant shifts were detected in abundance or community composition with altitude. The relative bacterial abundance increased at higher altitudes, which was related to increasing levels of soil organic matter and nutrients with altitude. Shifts in bacterial richness and diversity as well as community structure (comprised basically of Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes) significantly correlated with several environmental and soil chemical factors, especially soil pH. The site at the lowest altitude harbored the highest bacterial richness and diversity, although richness/diversity community properties did not show a monotonic decrease along the gradient. The relative size of fungal community also increased with altitude and its composition comprised Ascomycota, Basidiomycota, and Zygomycota. Changes in fungal richness/diversity and community structure were mainly governed by pH and C/N, respectively. The variation of the predominant bacterial and fungal classes over the altitudinal gradient was the result of the environmental and soil chemical factors prevailing at each site.

Effect of altitude and season on microbial activity, abundance and community structure in Alpine forest soils.

In the current context of climate change, the study of microbial communities along altitudinal gradients is especially useful. Only few studies considered altitude and season at the same time. We characterized four forest sites located in the Italian Alps, along an altitude gradient (545-2000 m a.s.l.), to evaluate the effect of altitude in spring and autumn on soil microbial properties. Each site in each season was characterized with regard to soil temperature, physicochemical properties, microbial activities (respiration, enzymes), community level physiological profiles (CLPP), microbial abundance and community structure (PLFA). Increased levels of soil organic matter (SOM) and nutrients were found at higher altitudes and in autumn, resulting in a significant increase of (soil dry-mass related) microbial activities and abundance at higher altitudes. Significant site- and season-specific effects were found for enzyme production. The significant interaction of the factors site and incubation temperature for soil microbial activities indicated differences in microbial communities and their responses to temperature among sites. CLPP revealed site-specific effects. Microbial community structure was influenced by altitudinal, seasonal and/or site-specific effects. Correlations demonstrated that altitude, and not season, was the main factor determining the changes in abiotic and biotic characteristics at the sites investigated.