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A novel method was developed for virus-like particle (VLP) extraction and characterization from biological soil crust (BSC) after microbial community reactivation. The method consisted of a single cell analysis by flow cytometry to monitor viable cells in BSC reactivated under controlled hydration, temperature, and light/dark exposure. Then, VLPs were extracted from reactivated BSCs, followed by viral DNA extraction and shotgun metagenomic analysis. The hydrated BSC under light/dark conditions showed the highest number of viable cells, and this condition was optimal for VLPs isolation. Taxonomic composition showed that families of the order of Caudovirales (Podoviridae, Myoviridae and Syphoviridiae) were the most abundant double strand DNA phages while Microviridiae were the most abundant single strand DNA phages. The isolated VLPs also carried sequences of relevant bacterial inhabiting soil. The functional categories of “phages, prophages, transposable elements, plasmids” and “clustering-base subsystem” were abundant (38 and 12%, respectively). All these data suggest viral predation as a key factor in shaping and maintaining bacterial diversity in the BSCs.

The aim of this work was to study the biological catalysts and possible substrate conversion routes in mesophilic dark fermentation reactors aimed at producing H2 from olive mill wastewater. Bacillus and Clostridium were the most abundant phylotypes during the rapid stage of H2 production. Chemical analyses combined with predictive functional profiling of the bacterial communities indicated that the lactate fermentation was the main H2-producing route. In fact, during the fermentation process, lactate and acetate were consumed, while H2 and butyrate were being produced. The fermentation process was rich in genes that encode enzymes for lactate generation from pyruvate. Lactate conversion to butyrate through the generation of pyruvate produced H2 through the recycling of electron carriers via the pyruvate ferredoxin oxydoreductase pathway. Overall, these findings showed the synergy among lactate-, acetate- and H2-producing bacteria, which complex interactions determine the H2 production routes in the bioreactors.

The open rock shelter of Yabelo in Ethiopia hosts diverse Holocene paintings of great cultural importance. The paintings are characterized by the presence of different mineral coatings, whose features have not been studied yet. Our goal was to understand whether different rock samples from the Yabelo paintings collected in close proximity may reveal coatings with different minerology and biology. Thus, elemental analyses combined with microscopic and molecular investigations were performed on two coatings, one whitish (sample 1) and one reddish (sample 2). Although both samples were dominated by heterotrophic bacteria, the two coatings showed distinct mineralogical and microbiological characteristics. Sample 1 contained higher amounts of Ca and P than sample 2, which was likely related to the presence of organic matter. Sample 1 hosted bacterial genera that are potentially involved in biomineralization processes, metal redox cycles and metal resistance. In contrast, sample 2 showed mainly pathogenic and commensal bacteria that are characteristic of animal and human microbiota, and other microorganisms that are involved in nitrogen and metal biogeochemical cycles. Overall, our results indicated that the bacterial communities were particular to the coating mineralogy, suggesting a potential role of the biological components in the crust genesis.

Biocrusts can be found in a wide array of habitats, where they provide important ecosystem services. These microbial associations are particularly important in High Arctic environments, where biocrust colonize the newly exposed barren soil after glacier retreat and significantly contribute to soil stabilization and nutrient cycling. Starting from incipient, structurally simple biolayers, they develop in complexity, increasing from the glacier terminus. Starting from a simple community structure, mainly constituted by cyanobacteria, heterotrophic bacteria and fungi immersed in a self-secreted extracellular polymeric matrix (cyanobacterial crusts), they later may recruit mosses and lichens (moss crusts and lichen crusts, respectively). The extracellular polymeric matrix protects the biocrust community from abiotic constraints, notably drought and freezing stress, from external physical harming factors, and from predation. The physicochemical characteristics of the extracellular matrix are related to several of its properties, such as its soil-stabilizing effect and water retention. We analysed the chemical (monosaccharidic composition) and macromolecular (molecular weight distribution) properties of the extracellular polymeric matrix of biocrusts with different morphologies collected in northwestern Spitsbergen, Norway. The uronic acid content and molecular weight (MW) distribution of the extracellular polysaccharidic matrices (EPMs) appeared in accordance with the developmental stages of the biocrusts. The MW distribution also showed significant differences between the samples, possibly reflecting differences in microbial enzymatic activities leading to the degradation of high-MW polymers into smaller compounds. The MW distribution profiles presented some important differences, reflecting differences in environmental conditions and, probably, the seasonal variance in microbial community composition that is known to characterize the environment examined in the present study.

Cyanobacteria are widespread prokaryotic organisms that represent feasible biotechnological tools to set up valid approaches to counteract desertification. Their peculiar physiological traits, and their resilience to abiotic stresses, allow their application on abiotically constrained soils to trigger their stabilization. A successful cyanobacteria inoculation results in the formation of cyanobacterial biocrusts, complex microbial communities characterized by tangled filament meshes imbued in a matrix of self-secreted extracellular polysaccharides (EPS) that keep loose sediments and aggregates firmly in place. However, the capability to form stable cyanobacterial biocrusts is not common to all the species, and a mix of factors can hamper the process, notably inoculum amount, and substrate characteristics. The aim of this work was to assess the influence of inoculum quantity and substrate granulometry on the physical stability of cyanobacterial biocrusts induced by inoculating the strain Leptolyngbya ohadii in a microcosm experiment, under laboratory conditions. After applying three different initial inoculum amounts on two different sand granulometries (medium and coarse sand), we assayed aggregate stability, physical stability and surface hydrophobicity on the resulting biocrusts during a 30-day incubation. Also, the features and the role of the EPS synthesized by L. ohadii were studied following their isolation, characterization, and direct application on the sand. The two EPS fractions produced by the strain, one more soluble and easily released in the surrounding medium (released polysaccharides, RPS) and one solidly attached to the filaments (glycocalyx EPS, G-EPS), were separately tested. Cyanobacterial biocrusts visibly formed in all the microcosms after 15 days. However, we observed a strong effect of sand granulometry in affecting aggregate stability and tensile strength, both of which appeared weaker on coarse sand. A higher amount of initial inoculum was necessary to produce stable biocrusts on coarse sand compared to medium sand. Also, we observed how the inoculation of EPS alone did not sort most of the significant effects that we detected by inoculating the whole culture, pointing at the importance of the action of the cyanobacterial filaments in soil conglomeration. However, a significant increase in physical stability was achieved by inoculating G-EPS on medium sand, suggesting the involvement of this fraction in biocrusts structuration. This work analyzes for the first time the effects of the variable grain size and inoculum amount in the achievement of physically stable biocrusts by cyanobacteria inoculation. The results that we obtained are useful in improving and optimizing the process of biomass preparation and dispersion for future indoor and outdoor studies.

© 2017, Springer International Publishing AG, part of Springer Nature. Aims: Biological soil crusts (biocrusts) are microbial communities commonly found in the upper layer of arid soils. These microorganisms release exopolysaccharides (EPS), which form the exopolymeric matrix (EPM), allowing them to bond soil particles together and survive long periods of dryness. The aim of this work is to develop methods for measuring metabolite retention by biocrust EPM and EPS. Methods: We report new methods for the investigation of metabolite sorption on biocrusts compared to the underlying unconsolidated subcrust fraction. A13C–labeled bacterial lysate metabolite mixture was incubated with biocrust, subcrust and biocrust-extracted EPS. Non-sorbed metabolites were extracted and analyzed by liquid chromatography/mass spectrometry. Results: This simple and rapid approach enabled the comparison of metabolite sorption on the biocrust EPM or EPS versus mineral sorption on the underlying soils. Our results suggest that the biocrust (and its extracted EPS) sorb more metabolites, especially amino acids and organic acids, than the underlying subcrust. Conclusions: This study demonstrates a useful method to highlight the essential role of biocrust (especially the EPM), which acts as a passive nutrient filter, sequestering metabolites released by microbes during wetting events. This may facilitate recovery of the community upon wetting and further enhance biocrust survival and nutrient retention.

Cyanobacteria are ubiquitous components of biocrust communities and the first colonizers of terrestrial ecosystems. They play multiple roles in the soil by fixing C and N and synthesizing exopolysaccharides, which increase soil fertility and water retention and improve soil structure and stability. Application of cyanobacteria as inoculants to promote biocrust development has been proposed as a novel biotechnological technique for restoring barren degraded areas and combating desertification processes in arid lands. However, previous to their widespread application under field conditions, research is needed to ensure the selection of the most suitable species. In this study, we inoculated two cyanobacterial species, Phormidium ambiguum (non N-fixing) and Scytonema javanicum (N-fixing), on different textured soils (from silt loam to sandy), and analyzed cyanobacteria biocrust development and evolution of physicochemical soil properties for three months under laboratory conditions. Cyanobacteria inoculation led to biocrust formation in all soil types. Scanning electron microscope (SEM) images showed contrasting structure of the biocrust induced by the two cyanobacteria. The one from P. ambiguum was characterized by thin filaments that enveloped soil particles and created a dense, entangled network, while the one from S. javanicum consisted of thicker filaments that grouped as bunches in between soil particles. Biocrust development, assessed by chlorophyll a content and crust spectral properties, was higher in S. javanicum-inoculated soils compared to P. ambiguum-inoculated soils. Either cyanobacteria inoculation did not increase soil hydrophobicity. S. javanicum promoted a higher increase in total organic C and total N content, while P. ambiguum was more effective in increasing total exopolysaccharide (EPS) content and soil penetration resistance. The effects of cyanobacteria inoculation also differed among soil types and the highest improvement in soil fertility compared to non-inoculated soils was found in sandy and silty soils, which originally had lowest fertility. On the whole, the improvement in soil fertility and stability supports the viability of using cyanobacteria to restore degraded arid soils.

Soil inoculation with cyanobacteria (cyanobacterization) is a biotechnological method widely studied to improve soil quality and productivity. During their growth on soil, cyanobacteria excrete exopolysaccharides (EPSs) which glue trichomes to soil particles, in a three-dimensional extracellular polymeric matrix. EPS productivity is an important screening parameter to select proficient inoculants and is affected by growth conditions and abiotic stresses. In this study, we evaluated the capability of the cyanobacterium Schizothrix cf. delicatissima AMPL0116 to form biocrusts when inoculated in sand microcosms under stressing conditions, and the characteristics of the synthesized polymeric matrix. In parallel, we evaluated the characteristics of exopolysaccharidic exudates of the strain when grown in liquid culture, under optimal growth setting. Our results pointed out at significant differences of the exopolymers produced in the two conditions in terms of monosaccharidic composition and molecular weight distribution, and proved the capability of S. cf. delicatissima AMPL0116 to form stable bioaggregates on sandy soils.

Soil inoculation with cyanobacteria to promote the formation of biocrusts is considered a potential eco-friendly method to counteract desertification spread in drylands. Research is needed to increase the number of proficient cyanobacterial strains, selected for their capability to survive in harsh conditions and to form stable biocrusts quickly. We hereby present a microcosm study to assess the capability of Leptolyngbya ohadii, native to the Negev Desert, to form biocrusts on sand collected in the same environment, during a three-month incubation period. Inoculation was carried out in sand-filled microcosms without nutrient addition and a limited water supply (equivalent to desert dew input). Parameters related to biocrusts growth and to their physico-chemical attributes were measured, and the exopolysaccharides (EPS) synthesized by the strain during biocrust formation were quantified and characterized. After 15 days of incubation, L. ohadii was able to form biocrusts with a thickness and a physical stability superior to other test strains of cyanobacteria, and typical of much older natural biocrusts. Biocrust characteristics were dependent on the synthesis of EPS, and on the capability to migrate in the sand, stabilizing sand aggregates at different locations within the microcosms. In contrast to other tested strains, L. ohadii produced compositionally complex EPS during the entire incubation period despite the lack of nutrients, producing biocrusts with an amphiphilic extracellular matrix, a character effective in conferring stability to sand aggregates, chelating nutrients and maintaining hydration. Overall, this study shows that L. ohadii is a promising inoculant that may be considered to promote the formation of biocrusts in natural desert settings.

Biological soil crusts (BSC) are soil-surface communities that represent a typical example of microbial adaptability to harsh environmental conditions. They improve soil fertility and promote plant growth, representing a valid putative tool to fight desertification processes. Various mechanisms allow them to withstand constraints such as drought, high solar irradiation and wind erosion. These organisms produce extracellular polymeric substances (EPS), excreted in response to the environmental characteristics, which are known to primarily contribute in many ways to the increase of constraint tolerance. While various features of these excreted molecules have been investigated in axenic bacterial cultures, a not definitive and relatively low number of studies come from the direct study of the entire matrix of microbial aggregates, with limitative information on how they contribute to microbial survival in extreme environments. The aim of this study was to investigate the microbial composition and the characteristics, both qualitative and quantitative, of the EPS extracted from BSC collected by several sites in Ny-Ǻlesund, Svalbard Island, Norway, in a landscape mostly characterized by the high arctic tundra, where environmental conditions are extreme and where BSC play a key role in creating a favorable habitat for plant growth after deglaciation processes. A proper method for extracting EPS directly from the crust samples was optimized. Exopolysaccharidic fractions were quantified and analyzed using ion-exchange chromatography (IEC) to determine the monosaccharidic composition. Size exclusion chromatography (SEC) was also used to determine the size distribution of the EPS fractions. Abundance of phototrophic microorganisms, which are known to primarily contribute to EPS excretion, was also evaluated in all the samples. Results underlined the complexity of the polysaccharidic fractions, displaying a high number of constituent sugars; the matrix was found to be constituted by two main fractions, a higher molecular weight (MW) fraction (apparent MW around 2 106 Da) and a lower molecular weight fraction (MW < 100 103 Da). Fluctuations of these results were observed along the sampling sites suggesting the influence of environmental factors and substrate characteristics on the formation of EPS matrix. This study presents novel data concerning EPS of BSCs matrix in cold environments and the results represent a starting point to deepen the knowledge on the mechanisms at the basis of the high tolerance showed by BSC microorganisms to extreme conditions. The knowledge of these mechanisms in different extreme environments (both hot and cold) can improve the outcome of the exploitation of these communities to increase the fertility and the stability of soils in constrained environments.