One versus two-stage codigestion of sugarcane vinasse and glycerol: Assessing combinations at mesophilic and (hyper) thermophilic conditions.

Sugarcane vinasse exits the distillation process at high temperatures, which may differ from the optimal temperatures for dark fermentation and anaerobic digestion. A 15 °C temperature increase, for example, stops sugarcane vinasse methane generation, making distillery vinasse digestion complicated. Conversely, in other aspects, co-digesting vinasse and glycerol has been proven to stabilize methane production from vinasse because of sulfate dilution. However, glycerol has not been tested to stabilize vinasse digestion under temperature changes. Thus, this study compared the effects of different temperature settings on the co-digestion of 10 g COD L-1 of vinasse and glycerol (50 %:50 % on a COD basis) in anaerobic fluidized bed reactors (AFBR), i.e., an acidogenic and a methanogenic one-stage AFBRs operated at 55, 60, and 65 °C, and two methanogenic AFBRs fed both with acidogenic effluent (one operated at room temperature (25 °C) and the other at 55, 60, and 65 °C). The co-digestion provided steady methane generation at all AFBRs, with methane production rates ranging from 2.27 to 2.93 L CH4 d-1 L-1, whether in one or two stages. A feature of this research was to unravel the black box of the role of sulfate in the digestion of sugarcane vinasse, which was rarely studied. Desulfovibrio was the primary genus degrading 1,3-propanediol into 3-hydroxypropanoate after genome sequencing. Phosphate acetyltransferase (EC: 2.3.1.8, K00625) and acetate kinase (EC: 2.7.2.1, K00925) genes were also found, suggesting propionate was metabolized. In practical aspects, regarding the two-stage systems, the thermophilic-mesophilic (acidogenic-methanogenic) configuration is best for extracting additional value-added products because 1,3-propanediol may be recovered at high yields with steady methane production at reduced energy expenditure in a reactor operated at room temperature. However, the one-stage design is best for methane generation per system volume since it remained stable with rising temperatures, and all systems presented similar methane production rates.

Interactive analysis of biosurfactants in fruit-waste fermentation samples using BioSurfDB and MEGAN.

Agroindustrial waste, such as fruit residues, are a renewable, abundant, low-cost, commonly-used carbon source. Biosurfactants are molecules of increasing interest due to their multifunctional properties, biodegradable nature and low toxicity, in comparison to synthetic surfactants. A better understanding of the associated microbial communities will aid prospecting for biosurfactant-producing microorganisms. In this study, six samples of fruit waste, from oranges, mangoes and mixed fruits, were subjected to autochthonous fermentation, so as to promote the growth of their associated microbiota, followed by short-read metagenomic sequencing. Using the DIAMOND+MEGAN analysis pipeline, taxonomic analysis shows that all six samples are dominated by Proteobacteria, in particular, a common core consisting of the genera Klebsiella, Enterobacter, Stenotrophomonas, Acinetobacter and Escherichia. Functional analysis indicates high similarity among samples and a significant number of reads map to genes that are involved in the biosynthesis of lipopeptide-class biosurfactants. Gene-centric analysis reveals Klebsiella as the main assignment for genes related to putisolvins biosynthesis. To simplify the interactive visualization and exploration of the surfactant-related genes in such samples, we have integrated the BiosurfDB classification into MEGAN and make this available. These results indicate that microbiota obtained from autochthonous fermentation have the genetic potential for biosynthesis of biosurfactants, suggesting that fruit wastes may provide a source of biosurfactant-producing microorganisms, with applications in the agricultural, chemical, food and pharmaceutical industries.

Marine associated microbial consortium applied to RBBR textile dye detoxification and decolorization: Combined approach and metatranscriptomic analysis.

The combination of different microorganisms and their metabolisms makes the use of microbial consortia in bioremediation processes a useful approach. In this sense, this study aimed at structuring and selecting a marine microbial consortium for Remazol Brilliant Blue R (RBBR) detoxification and decolorization. Experimental design was applied to improve the culture conditions, and metatranscriptomic analysis to understand the enzymatic pathways. A promising consortium composed of Mucor racemosus CBMAI 847, Marasmiellus sp. CBMAI 1062, Bacillus subtilis CBMAI 707, and Dietzia maris CBMAI 705 was selected. This consortium showed 52% of detoxification and 86% of decolorization in the validation assays after seven days of incubation in the presence of 500 ppm of RBBR. Reduction in RBBR color and toxicity were achieved by biosorption and microbial metabolisms. Metatranscriptomic data indicate that the consortium was able to decolorize and breakdown the RBBR molecule using a coordinated action of oxidases, oxygenases, and hydrolases. Epoxide hydrolases and glyoxalases expression could be associated with the decrease in toxicity. The efficiency of this marine microbial consortium suggests their use in bioremediation processes of textile effluents.

Exploring the genetic potential of a fosmid metagenomic library from an oil-impacted mangrove sediment for metabolism of aromatic compounds.

Aromatic hydrocarbons (AH) are widely distributed in nature, and many of them have been reported as relevant environmental pollutants and valuable carbon sources for different microorganisms. In this work, high-throughput sequencing of a metagenomic fosmid library was carried out to evaluate the functional and taxonomic diversity of genes involved in aromatic compounds degradation in oil-impacted mangrove sediments. In addition, activity-based approach and gas chromatography were used to assess the degradation potential of fosmid clones. Results indicated that AH degradation genes, such as monooxygenases and dioxygenases, were grouped into the following categories: anaerobic degradation of aromatic compounds (20.34%), metabolism of central aromatic intermediates (35.40%) and peripheral pathways for catabolism of aromatic compounds (22.56%). Taxonomic affiliation of genes related to aromatic compounds metabolism revealed the prevalence of the classes Alphaproteobacteria, Actinobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. Aromatic hydrocarbons (phenol, naphthalene, phenanthrene, pyrene and benzopyrene) were used as the only carbon source to screen clones with degradation potential. Of the 2500 clones tested, 48 showed some respiratory activity in at least one of the five carbon sources used. The hydrocarbon degradation ability of the top ten fosmid clones was confirmed by GC-MS. Further, annotation of assembled metagenomic fragments revealed ORFs corresponding to proteins and functional domains directly or indirectly involved in the aromatic compound metabolism, such as catechol 2,3-dioxygenase and ferredoxin oxidoreductase. Finally, these data suggest that the indigenous mangrove sediment microbiota developed essential mechanisms towards ecosystem remediation of petroleum hydrocarbon impact.

The metagenomic landscape of xenobiotics biodegradation in mangrove sediments.

Metagenomics is a powerful approach to study microorganisms present in any given environment and their potential to maintain and improve ecosystem health without the need of cultivating these microorganisms in the laboratory. In this study, we combined a cultivation-independent metagenomics approach with functional assays to identify the detoxification potential of microbial genes evaluating their potential to contribute to xenobiotics resistance in oil-impacted mangrove sediments. A metagenomic fosmid library containing 12,960 clones from highly contaminated mangrove sediment was used in this study. For assessment of metal resistance, clones were grown in culture medium with increasing concentrations of mercury. The analyses metagenomic library sequences revealed the presence of genes related to heavy metals and antibiotics resistance in the oil-impacted mangrove microbiome. The taxonomic profiling of these sequences suggests that at the genus level, Geobacter was the most abundant genus in our dataset. A functional screening assessment of the metagenomic library successfully detected 24 potential heavy metal tolerant clones, six of which were capable of growing with increased concentrations of mercury. The genetic characterization of selected clones allowed the detection of genes related to detoxification processes, such as chromate transport protein ChrA, haloacid dehalogenase-like hydrolase, lipopolysaccharide transport system, and 3-oxoacyl-[acyl-carrier-protein] reductase. Clones were capable of growing in medium containing increased concentrations of metals and antibiotics, but none manifested strong mercury removal from culture medium characteristic of mercuric reductase activity. These results suggest that resistance to xenobiotic stress varies greatly and that additional studies to elucidate the potential of metal biotransformation need to be carried out with the goal of improving bioremediation application.

Enzymatic potential of heterotrophic bacteria from a neutral copper mine drainage

Abstract Copper mine drainages are restricted environments that have been overlooked as sources of new biocatalysts for bioremediation and organic syntheses. Therefore, this study aimed to determine the enzymatic activities (esterase, epoxide hydrolase and monooxygenase) of 56 heterotrophic bacteria isolated from a neutral copper mine drainage (Sossego Mine, Canaã dos Carajás, Brazil). Hydrolase and monooxygenase activities were detected in 75% and 20% of the evaluated bacteria, respectively. Bacterial strains with good oxidative performance were also evaluated for biotransformation of organic sulfides. Fourteen strains with good enzymatic activity were identified by 16S rRNA gene sequencing, revealing the presence of three genera: Bacillus, Pseudomonas and Stenotrophomonas. The bacterial strains B. megaterium (SO5-4 and SO6-2) and Pseudomonas sp. (SO5-9) efficiently oxidized three different organic sulfides to their corresponding sulfoxides. In conclusion, this study revealed that neutral copper mine drainages are a promising source of biocatalysts for ester hydrolysis and sulfide oxidation/bioremediation. Furthermore, this is a novel biotechnological overview of the heterotrophic bacteria from a copper mine drainage, and this report may support further microbiological monitoring of this type of mine environment.

Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices

ABSTRACT Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.

Enzymatic potential of heterotrophic bacteria from a neutral copper mine drainage.

Copper mine drainages are restricted environments that have been overlooked as sources of new biocatalysts for bioremediation and organic syntheses. Therefore, this study aimed to determine the enzymatic activities (esterase, epoxide hydrolase and monooxygenase) of 56 heterotrophic bacteria isolated from a neutral copper mine drainage (Sossego Mine, Canaã dos Carajás, Brazil). Hydrolase and monooxygenase activities were detected in 75% and 20% of the evaluated bacteria, respectively. Bacterial strains with good oxidative performance were also evaluated for biotransformation of organic sulfides. Fourteen strains with good enzymatic activity were identified by 16S rRNA gene sequencing, revealing the presence of three genera: Bacillus, Pseudomonas and Stenotrophomonas. The bacterial strains B. megaterium (SO5-4 and SO6-2) and Pseudomonas sp. (SO5-9) efficiently oxidized three different organic sulfides to their corresponding sulfoxides. In conclusion, this study revealed that neutral copper mine drainages are a promising source of biocatalysts for ester hydrolysis and sulfide oxidation/bioremediation. Furthermore, this is a novel biotechnological overview of the heterotrophic bacteria from a copper mine drainage, and this report may support further microbiological monitoring of this type of mine environment.

Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices.

Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.

Bacterial diversity characterization in petroleum samples from Brazilian reservoirs / Caracterização da diversidade bacteriana em amostras de petróleo provenientes de reservatórios brasileiros

This study aimed at evaluating potential differences among the bacterial communities from formation water and oil samples originated from biodegraded and non-biodegraded Brazilian petroleum reservoirs by using a PCR-DGGE based approach. Environmental DNA was isolated and used in PCR reactions with bacterial primers, followed by separation of 16S rDNA fragments in the DGGE. PCR products were also cloned and sequenced, aiming at the taxonomic affiliation of the community members. The fingerprints obtained allowed the direct comparison among the bacterial communities from oil samples presenting distinct degrees of biodegradation, as well as between the communities of formation water and oil sample from the non-biodegraded reservoir. Very similar DGGE band profiles were observed for all samples, and the diversity of the predominant bacterial phylotypes was shown to be low. Cloning and sequencing results revealed major differences between formation water and oil samples from the non-biodegraded reservoir. Bacillus sp. and Halanaerobium sp. were shown to be the predominant components of the bacterial community from the formation water sample, whereas the oil sample also included Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. and Acidithiobacillus ferrooxidans. The PCR-DGGE technique, combined with cloning and sequencing of PCR products, revealed the presence of taxonomic groups not found previously in these samples when using cultivation-based methods and 16S rRNA gene library assembly, confirming the need of a polyphasic study in order to improve the knowledge of the extent of microbial diversity in such extreme environments.

Este estudo teve como objetivo comparar as comunidades bacterianas de amostras de água de formação e de óleo de reservatórios de petróleo brasileiros com diferentes graus de biodegradação usando a técnica de PCR-DGGE. O DNA ambiental foi isolado e empregado em reações de PCR com primers bacterianos, com subseqüente separação dos fragmentos de DNAr 16S em DGGE. Os produtos de PCR foram também clonados e seqüenciados, visando à afiliação taxonômica dos membros da comunidade. Os fingerprints obtidos permitiram a comparação direta entre as comunidades bacterianas das amostras de óleo com diferentes graus de biodegradação, assim como entre as comunidades da água de formação e do óleo do reservatório não biodegradado. Perfis de DGGE muito similares foram observados para todas as amostras, e a diversidade dos filotipos bacterianos predominantes mostrou-se baixa. Os resultados de clonagem e seqüenciamento revelaram diferenças mais significativas entre as amostras de água de formação e de óleo do reservatório não biodegradado. Bacillus sp. e Halanaerobium sp. mostraram-se os componentes predominantes da comunidade bacteriana da presente na amostra de água de formação, ao passo que a amostra de óleo incluiu também Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. e Acidithiobacillus ferrooxidans. A técnica de PCR-DGGE, combinada com clonagem e seqüenciamento dos produtos de PCR, revelou a presença de grupos taxonômicos não encontrados anteriormente nestas amostras quando métodos baseados em cultivo e na construção de bibliotecas de genes RNAr 16S foram utilizados, evidenciando a necessidade de um estudo polifásico a fim de contribuir para o conhecimento da diversidade microbiana em ambientes extremos como reservatórios de petróleo.