Methanotrophic Community Detected by DNA-SIP at Bertioga's Mangrove Area, Southeast Brazil.

Methanotrophic bacteria can use methane as sole carbon and energy source. Its importance in the environment is related to the mitigation of methane emissions from soil and water to the atmosphere. Brazilian mangroves are highly productive, have potential to methane production, and it is inferred that methanotrophic community is of great importance for this ecosystem. The scope of this study was to investigate the functional and taxonomic diversity of methanotrophic bacteria present in the anthropogenic impacted sediments from Bertioga´s mangrove (SP, Brazil). Sediment sample was cultivated with methane and the microbiota actively involved in methane oxidation was identified by DNA-based stable isotope probing (DNA-SIP) using methane as a labeled substrate. After 4 days (96 h) of incubation and consumption of 0.7 mmol of methane, the most active microorganisms were related to methanotrophs Methylomonas and Methylobacter as well as to methylotrophic Methylotenera, indicating a possible association of these bacterial groups within a methane-derived food chain in the Bertioga mangrove. The abundance of genera Methylomonas, able to couple methane oxidation to nitrate reduction, may indicate that under low dissolved oxygen tensions, some aerobic methanotrophs could shift to intraerobic methane oxidation to avoid oxygen starvation.

Sulfide-driven denitrification: detecting active microorganisms in fed-batch enrichment cultures by DNA stable isotope probing.

A microbial community was enriched in the anoxic compartment of a pilot-scale bioreactor that was operated for 180 days, fed with sewage and designed for organic matter, nitrogen and sulfide removal by coupling anaerobic digestion, nitrification and mixotrophic denitrification. Denitrification occurred with endogenous electron donors, mainly sulfide and residual organic matter, coming from the anaerobic compartment. The microorganisms involved in denitrification with sulfide as electron donor were identified by DNA-stable isotope probing with [U-13C]-labelled CO2 and NaHCO3. Complete denitrification occurred every two days, and the applied NO3-/S2- ratio was 1.6. Bacteria belonging to the Sulfurimonas denitrificans was identified as a chemoautotrophic denitrifier, and those related to Georgfuchisa toluolica, Geothrix fermentans and Ferritrophicum radicicola were most probably associated with heterotrophic denitrification using endogenous cells and/or intermediate metabolites. This study showed that DNA-SIP was a suitable technique to identify the active microbiota involved in sulfide-driven denitrification in a complex environment, which may contribute to improve design and operation of bioreactors aiming for carbon-nitrogen-sulfur removal.

Microbial community in a pilot-scale bioreactor promoting anaerobic digestion and sulfur-driven denitrification for domestic sewage treatment.

A pilot-scale reactor treating domestic sewage was operated to promote anaerobic digestion and denitrification using endogenous electron donors. While 55 % of organic matter was removed, nitrogen and sulfur showed a different dynamics during the operation. Pyrosequencing analysis clarified this behavior revealing that specific microbial communities inhabited the anaerobic (47.05 % of OTUs) and anoxic (31.39 % of OTUs) chambers. Analysis of 16S rRNA gene partial sequences obtained through pyrosequencing revealed a total of 1727 OTUs clustered at a 3 % distance cutoff. In the anaerobic chamber, microbial community was comprised of fermentative, syntrophic and sulfate-reducing bacteria. The majority of sequences were related to Aminobacterium and Syntrophorhabdus. In the anoxic chamber, the majority of sequences were related to mixotrophic and strictly autotrophic denitrifiers Arcobacter and Sulfuricurvum, respectively, both involved in sulfur-driven denitrification. These results show that pyrosequencing was a powerful tool to investigate the microbial panorama of a complex system, providing new insights to the improvement of the system.

Detection of Multiple Human Viruses, including Mpox, Using a Wastewater Surveillance Approach in Brazil.

Sewage surveillance can be used as an effective complementary tool for detecting pathogens in local communities, providing insights into emerging threats and aiding in the monitoring of outbreaks. In this study using qPCR and whole genomic sewage surveillance, we detected the Mpox virus along with other viruses, in municipal and hospital wastewaters in Belo Horizonte, Brazil over a 9-month period (from July 2022 until March 2023). MPXV DNA detection rates varied in our study, with 19.6% (11 out of 56 samples) detected through the hybrid capture method of whole-genome sequencing and 20% (12 out of 60 samples) through qPCR. In hospital wastewaters, the detection rate was higher, at 40% (12 out of 30 samples) compared to 13.3% (4 out of 30 samples) in municipal wastewaters. This variation could be attributed to the relatively low number of MPXV cases reported in the city, which ranged from 106 to 341 cases during the study period, and the dilution effects, given that each of the two wastewater treatment plants (WWTP) investigated serves approximately 1.1 million inhabitants. Additionally, nine other virus families were identified in both hospitals and municipal wastewaters, including Adenoviridade, Astroviridae, Caliciviridae, Picornaviridade, Polyomaviridae, Coronaviridae (which includes SARS-CoV-2), Herspesviridae, Papillomaviridae and Flaviviridae (notably including Dengue). These findings underscore the potential of genomic sewage surveillance as a robust public health tool for monitoring a wide range of viruses circulating in both community and hospitals environments, including MPXV.

Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process.

An anaerobic microbial community was enriched in a chemostat that was operated for more than 8 years with benzene and nitrate as electron acceptor. The coexistence of multiple species in the chemostat and the presence of a biofilm, led to the hypothesis that benzene-degrading species coexist in a syntrophic interaction, and that benzene can be degraded in syntrophy by consortia with various electron acceptors in the same culture. The benzene-degrading microorganisms were identified by DNA-stable isotope probing with [U-(13) C]-labelled benzene, and the effect of different electron donors and acceptors on benzene degradation was investigated. The degradation rate constant of benzene with nitrate (0.7 day(-1) ) was higher than reported previously. In the absence of nitrate, the microbial community was able to use sulfate, chlorate or ferric iron as electron acceptor. Bacteria belonging to the Peptococcaceae were identified as dominant benzene consumers, but also those related to Rhodocyclaceae and Burkholderiaceae were found to be associated with the anaerobic benzene degradation process. The benzene degradation activity in the chemostat was associated with microbial growth in biofilms. This, together with the inhibiting effect of hydrogen and the ability to degrade benzene with different electron acceptors, suggests that benzene was degraded via a syntrophic process.

Corrigendum: Higher Abundance of Sediment Methanogens and Methanotrophs Do Not Predict the Atmospheric Methane and Carbon Dioxide Flows in Eutrophic Tropical Freshwater Reservoirs.

[This corrects the article DOI: 10.3389/fmicb.2021.647921.].

Higher Abundance of Sediment Methanogens and Methanotrophs Do Not Predict the Atmospheric Methane and Carbon Dioxide Flows in Eutrophic Tropical Freshwater Reservoirs.

Freshwater reservoirs emit greenhouse gases (GHGs) such as methane (CH4) and carbon dioxide (CO2), contributing to global warming, mainly when impacted by untreated sewage and other anthropogenic sources. These gases can be produced by microbial organic carbon decomposition, but little is known about the microbiota and its participation in GHG production and consumption in these environments. In this paper we analyzed the sediment microbiota of three eutrophic tropical urban freshwater reservoirs, in different seasons and evaluated the correlations between microorganisms and the atmospheric CH4 and CO2 flows, also correlating them to limnological variables. Our results showed that deeper water columns promote high methanogen abundance, with predominance of acetoclastic Methanosaeta spp. and hydrogenotrophs Methanoregula spp. and Methanolinea spp. The aerobic methanotrophic community was affected by dissolved total carbon (DTC) and was dominated by Crenothrix spp. However, both relative abundance of the total methanogenic and aerobic methanotrophic communities in sediments were uncoupled to CH4 and CO2 flows. Network based approach showed that fermentative microbiota, including Leptolinea spp. and Longilinea spp., which produces substrates for methanogenesis, influence CH4 flows and was favored by anthropogenic pollution, such as untreated sewage loads. Additionally, less polluted conditions favored probable anaerobic methanotrophs such as Candidatus Bathyarchaeota, Sva0485, NC10, and MBG-D/DHVEG-1, which promoted lower gaseous flows, confirming the importance of sanitation improvement to reduce these flows in tropical urban freshwater reservoirs and their local and global warming impact.

Occurrence of methanogenic Archaea in highly polluted sediments of tropical Santos-São Vicente Estuary (São Paulo, Brazil).

Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos-São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30 degrees C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH(4)/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.

Development of a simple method to quantify fipronil and its intermediates in soil.

A simple and reproducible method was developed and validated for simultaneous quantification of the pesticide fipronil and its intermediates fipronil desulfinyl, fipronil sulfone and fipronil sulfide, in soil. The analytes were extracted by ultrasonic bath and the ratio of solvents (hexane/acetone), number and time of cycles were optimized by Box-Behnken design with a triplicate central point. The optimal extraction conditions were achieved through a response surface analysis. The clean-up step was conducted by cartridges of solid phase extraction (SPE) containing silica (Florisil®) and aluminum oxide. Gas chromatography with electron capture detection (GC-ECD) was employed for separating fipronil and its intermediates with a suitable resolution and runtime of 20 minutes. The best quantification was achieved with 1 : 1 (v/v) acetone/hexane and 2 ultrasound cycles of 15 minutes each. The recovery values were between 81 to 108%, with relative standard deviation (RSD) lower than 6%, with no effect of the used matrix. Analytical curves presented regression coefficients values above 0.9908 for a concentration range from 0.005 to 0.6 µg g-1. Limits of detection (LOD) from 0.002 to 0.006 µg g-1 and limits of quantification (LOQ) from 0.006 to 0.020 µg g-1 were reached for all analytes. This method can be used to monitor and quantify fipronil and its intermediates in soil.

Hexadecane biodegradation of high efficiency by bacterial isolates from Santos Basin sediments.

The aim of the study was the investigation of bacterial diversity from sediments collected at Santos Estuarine System, regarding to their abilities for hexadecane biotransformation. Hexadecane is a medium-chain linear alkane, considered as a model molecule for hydrocarbon biodegradation studies. It is a component from aliphatic fraction of crude petroleum, commonly related to environmental contamination by diesel oil. Santos Basin is an area with historical petroleum contamination. In the present work, sediment samples from this area were inoculated in artificial seawater (ASW), containing hexadecane as carbon source. Six bacterial isolates were selected as resistant to hexadecane. Chromatographic results showed biodegradation indexes above 97%. After 48 h of culture, five of them could degrade >80% of the initial hexadecane added. These isolates were characterized by 16S rDNA gene sequencing analysis. The following species were found: Bacillus amyloliquefaciens, Staphylococcus epidermidis, Micrococcus luteus, Nitratireductor aquimarinus, and Bacillus pumilus.

Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601(T.).

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601(T) have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601(T) is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601(T) are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601(T) and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601(T). Genes involved in cyclohexanol degradation were only found in strain K601(T). Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.

A strictly anaerobic betaproteobacterium Georgfuchsia toluolica gen. nov., sp. nov. degrades aromatic compounds with Fe(III), Mn(IV) or nitrate as an electron acceptor.

A bacterium (strain G5G6) that grows anaerobically with toluene was isolated from a polluted aquifer (Banisveld, the Netherlands). The bacterium uses Fe(III), Mn(IV) and nitrate as terminal electron acceptors for growth on aromatic compounds. The bacterium does not grow on sugars, lactate or acetate. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain G5G6 belonged to the Betaproteobacteria. Its closest, but only distantly related, cultured relative is Sterolibacterium denitrificans Chol-1S(T) (94.6% similarity of the 16S rRNA genes), a cholesterol-oxidizing, denitrifying bacterium. Strain G5G6 possesses the benzylsuccinate synthase A (bssA) gene encoding the alpha-subunit of Bss, which catalyzes the first step in anaerobic toluene degradation. The deduced BssA amino acid sequence is closely related to those of Azoarcus and Thauera species, which also belong to the Betaproteobacteria. Strain G5G6 is the first toluene-degrading, iron-reducing bacterium that does not belong to the Geobacteraceae within the Deltaproteobacteria. Based on phylogenetic and physiological comparison, strain G5G6 could not be assigned to a described species. Therefore, strain G5G6 (DSMZ 19032(T)=JCM 14632(T)) is a novel taxon of the Betaproteobacteria. We propose the name Georgfuchsia toluolica gen. nov., sp. nov.

Gas chromatographic methods for monitoring of wastewater chlorophenol degradation in anaerobic reactors.

Wastewater samples from an anaerobic reactor were extracted with hexane and derivatized with diazomethane (method 1) and with acetic anidride (method 2). Gas chromatography with electron-capture detection (ECD) was employed for separating the parent compound and intermediates trichlorophenols (TCP) and dichlorophenols (DCP) which originated from the penta chlorophenol (PCP) degradation process. The relations between concentrations of PCP, TCP and DCP areas were linear in the range of concentrations of 0.2 to 8 mg/L and 0.025 mg/L to 5 mg/L for methods 1 and 2, respectively. The repeatability of the extraction methods was satisfactory, with variation coefficients lower than 11%. For method 1, at the fortification level of 0.2 mg/L, recovery of PCP, TCP, and DCP was 112%, 74% and 45%, respectively. For method 2, the corresponding recovery values at the fortification level of 0.1 mg/L were 91%, 93% and 103%, respectively. Storage of the frozen samples did not alter their PCP determination properties. The chromatographic methods adapted for chlorophenol determination in wastewater were suitable with relatively simple manipulation techniques. The obtained results were reproducible and allowed identification of intermediates formed during the PCP degradation process.