Genomic, biochemical, and phylogenetic evaluation of bacteria isolated from deep-sea sediment harboring methane hydrates.

Over half of the organic carbon on Earth's surface is trapped in marine sediment as methane hydrates. Ocean warming causes hydrate dissociation and methane leakage to the water column, rendering the characterization of microbes from hydrate depositions a pressing matter. Through genomic, phylogenetic, and biochemical assays, we characterize the first microorganisms isolated from the Rio Grande Cone (Brazil), reservoir responsible for massive methane releases to the water column. From sediment harboring rich benthic communities, we obtained 43 strains of Brevibacillus sp., Paenibacillus sp. and groups of Bacillus sp. Methane-enriched samples yielded strains of the Pseudomonas fluorescens complex, exhibiting fluorescent siderophore production and broad multi-carbon catabolism. Genomic characterization of a novel Pseudomonas sp. strain indicated 32 genes not identified in the closest related type-species, including proteins involved with mercury resistance. Our results provide phylogenetic and genomic insights on the first bacterial isolates retrieved from a poorly explored region of the South Atlantic Ocean.

Biochemical features and early adhesion of marine Candida parapsilosis strains on high-density polyethylene.

AIMS: Plastic debris are constantly released into oceans where, due to weathering processes, they suffer fragmentation into micro- and nanoplastics. Diverse microbes often colonize these persisting fragments, contributing to their degradation. However, there are scarce reports regarding the biofilm formation of eukaryotic decomposing microorganisms on plastics. Here, we evaluated five yeast isolates from deep-sea sediment for catabolic properties and early adhesion ability on high-density polyethylene (HDPE). METHODS AND RESULTS: We assessed yeast catabolic features and adhesion ability on HDPE fragments subjected to abiotic weathering. Adhered cells were evaluated through Crystal Violet Assay, Scanning Electron Microscopy, Atomic Force Microscopy and Infrared Spectroscopy. Isolates were identified as Candida parapsilosis and exhibited wide catabolic capacity. Two isolates showed high adhesion ability on HDPE, consistently higher than the reference C. parapsilosis strain, despite an increase in fragment roughness due to weathering. Isolate Y5 displayed the most efficient colonization, with production of polysaccharides and lipids after 48 h of incubation. CONCLUSION: This work provides insights on catabolic metabolism and initial yeast-HDPE interactions of marine C. parapsilosis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings represent an essential contribution to the characterization of early interactions between deep-sea undescribed yeast strains and plastic pollutants found in oceans.

Biofilms of Pseudomonas and Lysinibacillus Marine Strains on High-Density Polyethylene.

Environmental pollution by plastic debris is estimated on a scale of 100 million metric tons, a portion of which is fragmented into micro- and nanoplastics. These fragments are often colonized by bacterial species in marine environments, possibly contributing to the biodegradation of such materials. However, further investigations are necessary to determine the impact of abiotic polymer weathering on biofilm adhesion, as well as the specific biofilm formation strategies employed by marine isolates. Here, we evaluate deep-sea sediment bacterial isolates for biofilm adhesion, extracellular matrix production, and polymer degradation ability. Our study focuses on high-density polyethylene (HDPE) fragments for their high durability and environmental persistence, subjecting fragments to abiotic weathering prior to bacterial colonization. Marine isolates identified as Pseudomonas sp. and Lysinibacillus sp. exhibited decreasing biofilm formation on weathered HDPE, especially over the first 24 h of incubation. This effect was countered by increased extracellular matrix production, likely improving cell adhesion to surfaces roughened by abiotic degradation. These adhesion strategies were contrasted with a reference Pseudomonas aeruginosa strain, which displayed high levels of biofilm formation on non-weathered HDPE and lower extracellular matrix production over the first 24 h of incubation. Furthermore, our results suggest that an increase in biofilm biomass correlated with changes to HDPE structure, indicating that these strains have a potential for biodegradation of plastic fragments. Therefore, this work provides a detailed account of biofilm formation strategies and bacteria-plastic interactions that represent crucial steps in the biodegradation of plastic fragments in marine environments.

Variation of the Prokaryotic and Eukaryotic Communities After Distinct Methods of Thermal Pretreatment of the Inoculum in Hydrogen-Production Reactors from Sugarcane Vinasse.

The aim of this study is to evaluate the effect of different thermal pretreatments of the inoculum on the diversity of the microbial community producing hydrogen from sugarcane vinasse. High-throughput sequencing of the 16S and 18S rRNA genes was performed. The reactor samples were also selected for the isolation of strict anaerobes. Decreased microbial diversity was observed with increasing pretreatment temperatures, with Firmicutes predominating: 90% to 97%. The highest abundance of Staphylococcus (7.9%) was found in pretreatment at 120 °C / 20 min at pH 6. The fungal analysis revealed a high prevalence of Candida (47%), Agaricomycetes, Pezizomycotina and Aspergillus in assays with higher H2 production (90° C / 10 min at pH 6). Three species of Clostridium were isolated: C. bifermentans, C. saccharoperbutylacetonicum and C. saccharobutylicum. The isolates were tested separately and in co-cultures for the production of hydrogen. Hydrogen-producing capacity by co-culture of Clostridium species was increased by 18%. Knowing microorganisms and understanding the interaction between eukaryotes and prokaryotes is essential to obtain strategies for biotransformation of vinasse for the production of bioenergy.

Microbiota profile in mesophilic biodigestion of sugarcane vinasse in batch reactors.

The vinasse is a residue of ethanol production with the potential for methane production, requiring an allochthonous inoculum. Several microorganisms act in the different phases of anaerobic digestion, and the identification of these microbial communities is essential to optimize the process. The characterization of the microbiota involved in the biodigestion of vinasse was observed in the initial stage (IS), at the peak of methane production (MS) and the end of the process (FS) of the best performance assay by high-throughput sequencing. The highest methane production was 0.78 mmolCH4.gVS.h-1 at 243.7 h in the substrate/inoculum ratio of 1.7, with consumption partial of acetic, propionic and isobutyric acids and an 82% reduction of chemical oxygen demand. High microbial diversity was found. The genera Clostridium, Acinetobacter, Candidatus Cloacamonas, Bacteroides, Syntrophomonas, Kosmotoga, the family Porphyromonadaceae and the class Bacteroidia were the most abundant in the maximum methane production. Methane production was driven by Methanobacterium and Methanosaeta, suggesting the metabolic pathways used were hydrogenotrophic and acetoclastic.

Mollusk microbiota shift during Angiostrongylus cantonensis infection in the freshwater snail Biomphalaria glabrata and the terrestrial slug Phillocaulis soleiformis.

In the present work, we reported for the first time the microbiome from Phyllocaulis soleiformis and Biomphalaria glabrata assessed using high-throughput DNA sequencing pre- and post-infection with the helminth parasite Angiostrongylus cantonensis. B. glabrata and P. soleiformis were experimentally infected with A. cantonensis. Fecal DNAs from control and infected groups were extracted and subjected to 16S rRNA high-throughput sequencing survey. No significant differences were found in the alpha diversity indexes in Phyllocaulis and Biomphalaria experiments independently. PCoA analysis using the unweighted UniFrac measures showed that both microbiotas behaved differently depending on the host. In Biomphalaria microbiota, control and infected groups were significantly different (p = 0.0219), while Phyllocaulis samples were not (p = 0.5190). The microbiome of P. soleiformis infected with A. cantonensis showed a significant decrease of Sphingobacterium and a substantial increase of Cellvibrio when compared to a control group. The microbiome of B. glabrata infected with A. cantonensis showed a significant decline in the abundance of Flavobacterium, Fluviicola, Nitrospira, Vogesella and an OTU belonging to the family Comamonadaceae, and a significant increase of Uliginosibacterium and an OTU belonging to the family Weeksellaceae when compared to a control group. Overall, the microbiome data reported here provided valuable information with regard to the diversity of bacterial communities that comprise the gut microbiome of gastropods. Furthermore, we report here the effect of the infection of the helminth A. cantonensis in the ratio and distribution of the fecal microbiome of the snails. Further studies are highly valuable in order to better understand those interactions by comparing different microbiome profiles and mollusk models. By now, we anticipate that ecological studies will take significant advantage of these advances, particularly concerning improving our understanding of helminth-microbiome-host interactions.

UV-Surface Treatment of Fungal Resistant Polyether Polyurethane Film-Induced Growth of Entomopathogenic Fungi.

Synthetic polymers are the cause of some major environmental impacts due to their low degradation rates. Polyurethanes (PU) are widely used synthetic polymers, and their growing use in industry has produced an increase in plastic waste. A commercial polyether-based thermoplastic PU with hydrolytic stability and fungus resistance was only attacked by an entomopathogenic fungus, Metarhiziumanisopliae, when the films were pre-treated with Ultraviolet (UV) irradiation in the presence of reactive atmospheres. Water contact angle, Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR), scanning electron microscopy (SEM), and profilometer measurements were mainly used for analysis. Permanent hydrophilic PU films were produced by the UV-assisted treatments. Pristine polyether PU films incubated for 10, 30, and 60 days did not show any indication of fungal growth. On the contrary, when using oxygen in the UV pre-treatment a layer of fungi spores covered the sample, indicating a great adherence of the microorganisms to the polymer. However, if acrylic acid vapors were used during the UV pre-treatment, a visible attack by the entomopathogenic fungi was observed. SEM and FTIR-ATR data showed clear evidence of fungal development: growth and ramifications of hyphae on the polymer surface with the increase in UV pre-treatment time and fungus incubation time. The results indicated that the simple UV surface activation process has proven to be a promising alternative for polyether PU waste management.

Exploratory analysis of the microbial community profile of the municipal solid waste leachate treatment system: A case study.

Studies on the degradation dynamics of landfill leachate indicate that the microbial community profile is a valuable and sensitive tool for landfill monitoring programs. Although knowledge about the microbial community can improve the efficiency of leachate treatment systems, little is known about the microbial profile changes that occur throughout the leachate attenuation process. In the present work, an exploratory analysis of the microbial community profile of the MSW leachate treatment system in the municipality of Osório (Brazil) was conducted. In this way, a comprehensive analysis of chemical parameters, isotopic signature and microbial profile data were applied to monitor the changes in the structure of the microbial community throughout the leachate attenuation process and to describe the relationship between the microbial community structure and the attenuation of chemical and isotopic parameters. From data analysis, it was possible to assess the microbial community structure and relate it to the attenuation of chemical and isotopic parameters. Based on massive parallel 16S rRNA gene sequencing, it was possible to observe that each leachate treatment unit has a specific microbial consortium, reflecting the adaptation of different microorganisms to changes in leachate characteristics throughout treatment. From our results, we concluded that the structure of the microbial community is sensitive to the leachate composition and can be applied to study the municipal solid waste management system.

Prokaryotic and microeukaryotic communities in an experimental rice plantation under long-term use of pesticides.

Conventional agricultural practices, such as rice plantations, often contaminate the soil and water with xenobiotics. Here we evaluated the microbiota composition in experimental rice planting with a record of prolonged pesticide use, using 16S and 18S rRNA amplicon sequencing. We investigated four components of a complete agricultural system: affluent water (A), rice rhizosphere soil (R), sediment from a storage pond (S), and effluent (E) water (drained from the storage pond). Despite the short spatial distance between our sites, the beta diversity analysis of bacterial communities showed two well-defined clusters, separating the water and sediment/rhizosphere samples; rhizosphere and sediment were richer while the effluent was less diverse. Overall, the site with the highest evenness was the rhizosphere. Unlike the bacterial communities, Shannon diversity of microeukaryotes was significantly different between A and E. The effluent presented the lowest values for all ecological indexes tested and differed significantly from all sampled sites, except on evenness. When mapped the metabolic pathways, genes corresponding to the degradation of aromatic compounds, including genes related to pesticide degradation, were identified. The most abundant genes were related to the degradation of benzoate. Our results indicate that the effluent is a selective environment for fungi. Interestingly, the overall fungal diversity was higher in the affluent, the water that reached the system before pesticide application, and where the prokaryotic diversity was the lowest. The affluent and effluent seem to have the lowest environmental quality, given the presence of bacteria genera previously recorded in environments with high concentrations of pesticide residues. The microbiota, environmental characteristics, and pesticide residues should be further studied and try to elucidate the potential for pesticide degradation by natural consortia. Thus, extensive comparative studies are needed to clarify the microbial composition, diversity, and functioning of rice cultivation environments, and how pesticide use changes may reflect differences in microbial structure.

Determination of the Nutritional Value of Diet Containing Bacillus subtilis Hydrolyzed Feather Meal in Adult Dogs.

Feathers are naturally made up of non-digestible proteins. Under thermal processing, total tract digestibility can be partially improved. Furthermore, Bacillus subtilis (Bs) has shown a hydrolytic effect In vitro. Then, a Bs FTC01 was selected to hydrolyze enough feathers to produce a meal, and then test the quality and inclusion in the dog's diet to measure the apparent total tract digestibility coefficient (ATTDC) in vivo and the microorganism's ability to survive in the gastrointestinal tract. A basal diet was added with 9.09% hydrolyzed Bs feather meal (HFMBs) or 9.09% thermally hydrolyzed feather meal (HFMT). Nine adult dogs were randomized into two 10-day blocks and fed different diets. Microbial counts were performed on feather meal, diets and feces. The Bs was less effective in digesting the feathers, which reduced the ATTDC of dry matter, crude protein, energy and increased the production of fecal DM, but the fecal score was maintained (p > 0.05). The digestible energy of HFMT and HFMBs was 18,590 J/kg and 9196 J/kg, respectively. Bacillus subtilis showed limitation to digest feather in large scale, but the resistance of Bs to digestion was observed since it grown on feces culture.

Oral Phenotype and Salivary Microbiome of Individuals With Papillon-Lefèvre Syndrome.

Papillon-Lefèvre syndrome (PLS) is an autosomal recessive rare disease, main characteristics of which include palmoplantar hyperkeratosis and premature edentulism due to advanced periodontitis (formerly aggressive periodontitis). This study aimed to characterize the oral phenotype, including salivary parameters, and the salivary microbiome of three PLS sisters, comparatively. Two sisters were toothless (PLSTL1 and PLSTL2), and one sister had most of the teeth in the oral cavity (PLST). Total DNA was extracted from the unstimulated saliva, and the amplicon sequencing of the 16S rRNA gene fragment was performed in an Ion PGM platform. The amplicon sequence variants (ASVs) were obtained using the DADA2 pipeline, and the taxonomy was assigned using the SILVA v.138. The main phenotypic characteristics of PLS were bone loss and premature loss of primary and permanent dentition. The PLST sister presented advanced periodontitis with gingival bleeding and suppuration, corresponding to the advanced periodontitis as a manifestation of systemic disease, stage IV, grade C. All three PLS sisters presented hyposalivation as a possible secondary outcome of the syndrome. Interestingly, PLST salivary microbiota was dominated by the uncultured bacteria Bacterioidales (F0058), Fusobacterium, Treponema, and Sulfophobococcus (Archaea domain). Streptococcus, Haemophilus, and Caldivirga (Archaea) dominated the microbiome of the PLSTL1 sister, while the PLSTL2 had higher abundances of Lactobacillus and Porphyromonas. This study was the first to show a high abundance of organisms belonging to the Archaea domain comprising a core microbiome in human saliva. In conclusion, a PLST individual does have a microbiota different from that of the periodontitis' aggressiveness previously recognized. Due to an ineffective cathepsin C, the impairment of neutrophils probably provided a favorable environment for the PLS microbiome. The interactions of Bacteroidales F0058, Caldivirga, and Sulfophobococcus with the microbial consortium of PLS deserves future investigation. Traditional periodontal therapy is not efficient in PLS patients. Unraveling the PLS microbiome is essential in searching for appropriate treatment and avoiding early tooth loss.

Presence of Archaea in dental caries biofilms.

OBJECTIVE: Although the prevalence and functions associated with members of Bacteria are well known in dental caries, the role of Archaea in cariogenic biofilms has not been studied yet. DESIGN: To detect the presence of Archaea in dental caries, a triplicate of carious dentine samples and duplicate of supragingival biofilms were collected, total microbial DNA was extracted and the composition of the microbiota was investigated. Total DNA was submitted to 16S rRNA gene amplification using universal prokaryotic primers; amplicons were sequenced by high-throughput DNA sequencing. As a second strategy to detect Archaea, a representative sample of caries was chosen and other PCR reactions were performed using specific primers targeting the archaeal 16S rRNA gene; amplicons were cloned and sequenced. Annotation of sequences was performed using SILVA database and the relative abundance of genus level OTUs was calculated. RESULTS: The high-throughput sequencing method detected archaeal sequences in all samples (identified as group I.1c of the phylum Thaumarchaeota), although in a very low abundance (≤0.03 % of the total sequences). For the second strategy, 14 archaeal clones were detected, with an OTU affiliated to Methanocella clade, and another one affiliated to group I.1b of the phylum Thaumarchaeota. CONCLUSIONS: Archaeal sequences were detected in dental caries and biofilms from surfaces without caries lesions. DNA sequences of Thaumarchaeota were also identified, showing that overall archaeal diversity in the human oral cavity could be currently underestimated and not restricted to methanogens.

Adaption of microbial communities to the hostile environment in the Doce River after the collapse of two iron ore tailing dams.

In November 2015, two iron ore tailing dams collapsed in the city of Mariana, Brazil. The dams' collapse generated a wave of approximately 50 million m3 of a mixture of mining waste and water. It was a major environmental tragedy in Brazilian history, which damaged rivers, and cities 660 km away in the Doce River basin until it reached the ocean coast. Shortly after the incident, several reports informed that the concentration of metals in the water was above acceptable legal limits under Brazilian laws. Here the microbial communities in samples of water, mud, foam, and rhizosphere of Eichhornia from Doce River were analyzed for 16S and 18S rRNA-based amplicon sequencing, along with microbial isolation, chemical and mineralogical analyses. Samples were collected one month and thirteen months after the collapse. Prokaryotic communities from mud shifted drastically over time (33% Bray-Curtis similarity), while water samples were more similar (63% Bray-Curtis similarity) in the same period. After 12 months, mud samples remained with high levels of heavy metals and a reduction in the diversity of microeukaryotes was detected. Amoebozoans increased in mud samples, reaching 49% of microeukaryote abundance, with Discosea and Lobosa groups being the most abundant. The microbial communities' structure in mud samples changed adapting to the new environment condition. The characterization of microbial communities and metal-tolerant organisms from such impacted environments is essential for understanding the ecological consequences of massive anthropogenic impacts and strategies for the restoration of contaminated sites such as the Doce River.

Biodegradation improvement of poly(3-hydroxy-butyrate) films by entomopathogenic fungi and UV-assisted surface functionalization.

Ultraviolet (UV)-assisted surface modification in the presence of oxygen was used as initial step to achieve controlled degradation of poly(3-hydroxy-butyrate), PHB, films by entomopathogenic fungi. Treated surfaces were investigated by surface analysis techniques (water contact angle, Fourier Transformed Infrared Spectroscopy in Attenuated Total Reflectance mode, X-ray Photoelectron Spectroscopy, Near-edge X-ray Absorption Fine Structure, Gel Permeation Chromatography, Optical Microscopy, Scanning Electron Microscopy, and weight loss). After the UV-assisted treatments, new carbonyl groups in new chemical environments were detected by XPS and NEXAFS spectroscopy. The oxidizing atmosphere did not allow the formation of CC bonds, indicating that Norrish Type II mechanism is suppressed during or by the treatments. The higher hydrophilicity and concentration of oxygenated functional groups at the surface of the treated films possibly improved the biodegradation of the films. It was observed a clear increase in the growth of this fungus when oxygenated groups were grafted on the polymers surfaces. This simple methodology can be used to improve and control the degradation rate of PHB films in applications that require a controllable degradation rate.