Microbial Ecology of Artisanal Feta and Kefalograviera Cheeses, Part I: Bacterial Community and Its Functional Characteristics with Focus on Lactic Acid Bacteria as Determined by Culture-Dependent Methods and Phenotype Microarrays

Artisanal cheesemaking is still performed using practices and conditions derived from tradition. Feta and Kefalograviera cheeses are very popular in Greece and have met worldwide commercial success. However, there is a lack of knowledge regarding their lactic acid microecosystem composition and species dynamics during ripening. Thus, the aim of the present study was to assess the microecosystem as well as the autochthonous lactic acid microbiota during the ripening of artisanal Feta and Kefalograviera cheeses. For that purpose, raw sheep’s milk intended for cheesemaking, as well as Feta and Kefalograviera cheeses during early and late ripening were analyzed, and the lactic acid microbiota was identified using the classical phenotypic approach, clustering with PCR-RAPD and identification with sequencing of the 16S-rRNA gene, as well as with the Biolog GEN III microplates. In addition, the functional properties of the bacterial community were evaluated using the Biolog EcoPlates, which consists of 31 different carbon sources. In general, concordance between the techniques used was achieved. The most frequently isolated species from raw sheep’s milk were Enteroroccus faecium, Lactiplantibacillus plantarum and Pediococcus pentosaceus. The microecosystem of Feta cheese in the early ripening stage was dominated by Lp. plantarum and E. faecium, whereas, in late ripening, the microecosystem was dominated by Weissella paramesenteroides. The microecosystem of Kefalograviera cheese in the early ripening stage was dominated by Levilactobacillus brevis and E. faecium, and in late ripening by W. paramesenteroides and E. faecium. Finally, Carbohydrates was the main carbon source category that metabolized by all microbial communities, but the extent of their utilization was varied. Kefalograviera samples, especially at early ripening, demonstrated higher metabolic activity compared to Feta cheese. However, dominating species within microbial communities of the cheese samples were not significantly different.

Pollution-Induced Tolerance of Soil Bacterial Communities to Oxytetracycline-Spiked Manure

The use of manure as a fertilizer is a common agricultural practice that can improve soil physicochemical and biological properties. However, antibiotics and their metabolites are often present, leading to the adaptation of soil bacterial communities to their presence. The aim of this study was to assess the effects of the extensively used, broad-spectrum antibiotic oxytetracycline on soil microbial community adaptation using a pollution-induced community tolerance assay. Manure-amended soil was spiked with oxytetracycline (0, 2, 20, 60, 150, and 500 mg kg−1) three times every ten days in the selection phase. The detection phase was conducted in Biolog EcoPlates with a second oxytetracycline exposure (0, 5, 20, 40, 60, and 100 mg L−1). All treatments demonstrated decreased metabolic activity after exposure to ≥ 5 mg L−1 oxytetracycline during the detection phase. Meanwhile, a significant increase in tolerance was observed following exposure to ≥ 20 mg oxytetracycline per kg soil during the selection phase. Therefore, the pollution-induced community tolerance approach with Biolog EcoPlates was a useful system for the detection of antibiotic selection pressures on soil bacterial communities. It is important to properly manage animal waste before their application to the soil to reduce the occurrence of antibiotic-resistance in the environment.

Root-Associated Bacteria Community Characteristics of Antarctic Plants: Deschampsia antarctica and Colobanthus quitensis—a Comparison

Colobanthus quitensis (Kunth) Bartl. and Deschampsia antarctica Desv. are the only Magnoliophyta to naturally colonize the Antarctic region. The reason for their sole presence in Antarctica is still debated as there is no definitive consensus on how only two unrelated flowering plants managed to establish breeding populations in this part of the world. In this study, we have explored and compared the rhizosphere and root-endosphere dwelling microbial community of C. quitensis and D. antarctica specimens sampled in maritime Antarctica from sites displaying contrasting edaphic characteristics. Bacterial phylogenetic diversity (high-throughput 16S rRNA gene fragment targeted sequencing) and microbial metabolic activity (Biolog EcoPlates) with a geochemical soil background were assessed. Gathered data showed that the microbiome of C. quitensis root system was mostly site-dependent, displaying different characteristics in each of the examined locations. This plant tolerated an active bacterial community only in severe conditions (salt stress and nutrient deprivation), while in other more favorable circumstances, it restricted microbial activity, with a possibility of microbivory-based nutrient acquisition. The microbial communities of D. antarctica showed a high degree of similarity between samples within a particular rhizocompartment. The grass’ endosphere was significantly enriched in plant beneficial taxa of the family Rhizobiaceae, which displayed obligatory endophyte characteristics, suggesting that at least part of this community is transmitted vertically. Ultimately, the ecological success of C. quitensis and D. antarctica in Antarctica might be largely attributed to their associations and management of root-associated microbiota.

Functional Metabolic Diversity of Bacterioplankton in Maritime Antarctic Lakes

A summer survey was conducted on the bacterioplankton communities of seven lakes from Byers Peninsula (Maritime Antarctica), differing in trophic and morphological characteristics. Predictions of the metabolic capabilities of these communities were performed with FAPROTAX using 16S rRNA sequencing data. The versatility for metabolizing carbon sources was also assessed in three of the lakes using Biolog Ecoplates. Relevant differences among lakes and within lake depths were observed. A total of 23 metabolic activities associated to the main biogeochemical cycles were foreseen, namely, carbon (11), nitrogen (4), sulfur (5), iron (2), and hydrogen (1). The aerobic metabolisms dominated, although anaerobic respiration was also relevant near the lakes’ bottom as well as in shallow eutrophic lakes with higher nutrient and organic matter contents. Capacity for using carbon sources further than those derived from the fresh autochthonous primary production was detected. Clustering of the lakes based on metabolic capabilities of their microbial communities was determined by their trophic status, with functional diversity increasing with trophic status. Data were also examined using a co-occurrence network approach, indicating that the lakes and their catchments have to be perceived as connected and interacting macrosystems, where either stochastic or deterministic mechanisms for the assembling of communities may occur depending on the lake’s isolation. The hydrological processes within catchments and the potential metabolic plasticity of these biological communities must be considered for future climate scenarios in the region, which may extend the growing season and increase biomass circulation.

Analysis of Bacterial Community level Physiological Profiling on the Fermentation of Traditional Pliek u using BIOLOG EcoPlates

Pliek u is an Acehnese traditional condiment made from fermented coconut (Cocos nucifera) endosperm. The traditional pliek u fermentation process typically involves a diverse bacterial community. This research aimed to discover the physiological profile of the bacterial community diversity in pliek u fermentation. BIOLOGTM EcoPlates was used to obtain the physiological functions of the bacterial community during the pliek u fermentation process. The bacteria were then isolated from EcoPlate substrate to determine the predominant microorganism. Results from the analysis showed that the value of the Average Well Colour Development (AWCD) increased during the EcoPlates incubation period. The AWCD values in sample IV were higher than the AWCD values in samples I, II, and III. PCA analysis showed that the use of EcoPlate substrate by the bacterial community at the beginning of the fermentation was correlated with the substrate groups of carbohydrate and polymer, and with the substrate groups of carbohydrate and the amino acid at the end of the fermentation. The phylogenetic analysis showed that EC1 had a close relation with Pseudomonas azotoformans strain NBRC, while EC3 had a close relation with Psedomonas lundensis strain ATCC 49968. In conclusion, there were changes in the use of EcoPlate substrate and the activities of the bacterial community during the pliek u fermentation process.

The ecological effects of land-applying municipal biosolids on nitrogen-fixing bacteria

The effects of municipal biosolids on nitrogen-fixing bacteria were assessed in a three month soil incubation study. Treatments included reference agricultural soils, soil amended with municipal biosolids or manure, and biosolids without soil. Nitrogen-fixation rates in reference and manure-amended soil were similar, and lower than in biosolids treatments; respiration rates showed similar trends. At test termination there was no difference between soil treatments for nitrogen-fixation, but some enhanced respiration in the biosolids-amended soils. Community structure was assessed using Biolog EcoPlates™ and denaturing gradient gel electrphoresis with a nitrogen-fixing gene (nifH). EcoPlate™ carbon utilization patterns corresponded with activity measures, with no difference among soil treatments at test termination. Nitrogen-fixing gene patterns showed a potential shift in community structure of biosolids-amended soils three months post-amendment. In general, the effects on the activity and structure of nitrogen-fixing communities were largely temporary; however, this study evaluated a one-time biosolids application. The potential for cumulative effects requires further investigation.

The ecological effects of land-applying municipal biosolids on nitrogen-fixing bacteria

The effects of municipal biosolids on nitrogen-fixing bacteria were assessed in a three month soil incubation study. Treatments included reference agricultural soils, soil amended with municipal biosolids or manure, and biosolids without soil. Nitrogen-fixation rates in reference and manure-amended soil were similar, and lower than in biosolids treatments; respiration rates showed similar trends. At test termination there was no difference between soil treatments for nitrogen-fixation, but some enhanced respiration in the biosolids-amended soils. Community structure was assessed using Biolog EcoPlates™ and denaturing gradient gel electrphoresis with a nitrogen-fixing gene (nifH). EcoPlate™ carbon utilization patterns corresponded with activity measures, with no difference among soil treatments at test termination. Nitrogen-fixing gene patterns showed a potential shift in community structure of biosolids-amended soils three months post-amendment. In general, the effects on the activity and structure of nitrogen-fixing communities were largely temporary; however, this study evaluated a one-time biosolids application. The potential for cumulative effects requires further investigation.

Understanding the Shift in the Microbiome of Composts That Are Optimized for a Better Fit-for-Purpose in Growing Media

Three characteristics are considered key for optimal use of composts in growing media: maturity, pH and organic matter content. Maturation is a critical step in the processing of composts contributing to compost quality. Blending of composts with chopped heath biomass, sieving out the larger fraction of composts and acidification of composts by adding elemental sulfur may be used either to increase organic matter content or to reduce pH for a better fit in growing media. While several studies have shown the effectiveness of these treatments to improve the use of composts in growing media, the effect of these treatments on the compost microbiome has merely been assessed before. In the present study, five immature composts were allowed to mature, and were subsequently acidified, blended or sieved. Bacterial and fungal communities of the composts were characterized and quantified using 16S rRNA and ITS2 gene metabarcoding and phospholipid fatty acid analysis. Metabolic biodiversity and activity were analyzed using Biolog EcoPlates. Compost batch was shown to be more important than maturation or optimization treatments to determine the compost microbiome. Compost maturation increased microbial diversity and favored beneficial microorganisms, which may be positive for the use of composts in growing media. Blending of composts increased microbial diversity, metabolic diversity, and metabolic activity, which may have a positive effect in growing media. Blending may be used to modify the microbiome to a certain degree in order to optimize microbiological characteristics. Acidification caused a decrease in bacterial diversity and microbial activity, which may be negative for the use in growing media, although the changes are limited. Sieving had limited effect on the microbiome of composts. Because of the limited effect on the microbiome, sieving of composts may be used flexible to improve (bio)chemical characteristics. This is the first study to assess the effects of maturation and optimization treatments to either increase organic matter content or lower pH in composts on the compost microbiome.

Graphene-Based Nanomaterials Modulate Internal Biofilm Interactions and Microbial Diversity

Graphene-based nanomaterials (GBMs), such as graphene oxide (GO) and reduced graphene oxide (rGO), possess unique properties triggering high expectations for the development of new technological applications and are forecasted to be produced at industrial-scale. This raises the question of potential adverse outcomes on living organisms and especially toward microorganisms constituting the basis of the trophic chain in ecosystems. However, investigations on GBMs toxicity were performed on various microorganisms using single species that are helpful to determine toxicity mechanisms but fail to predict the consequences of the observed effects at a larger organization scale. Thus, this study focuses on the ecotoxicological assessment of GO and rGO toward a biofilm composed of the diatom Nitzschia palea associated to a bacterial consortium. After 48 and 144 h of exposure to these GBMs at 0, 0.1, 1, and 10 mg.L−1, their effects on the diatom physiology, the structure, and the metabolism of bacterial communities were measured through the use of flow cytometry, 16S amplicon sequencing, and Biolog ecoplates, respectively. The exposure to both of these GBMs stimulated the diatom growth. Besides, GO exerted strong bacterial growth inhibition as from 1 mg.L−1, influenced the taxonomic composition of diatom-associated bacterial consortium, and increased transiently the bacterial activity related to carbon cycling, with weak toxicity toward the diatom. On the contrary, rGO was shown to exert a weaker toxicity toward the bacterial consortium, whereas it influenced more strongly the diatom physiology. When compared to the results from the literature using single species tests, our study suggests that diatoms benefited from diatom-bacteria interactions and that the biofilm was able to maintain or recover its carbon-related metabolic activities when exposed to GBMs.