Thymol as Starting Material for the Development of a Biobased Material with Enhanced Antimicrobial Activity: Synthesis, Characterization, and Potential Application.

A biobased material, polythymol (PTF), was prepared using thymol, a monoterpene obtained from the essential oil of Thymus vulgaris (Lamiaceae), as a starting material with the aim of enhancing the antimicrobial properties of this natural product. Initially, different processes were performed in order to optimize the reaction conditions to obtain a macromolecule with a high purity and yield. PTF was characterized using different techniques, such as NMR, infrared, UV-Vis, and thermogravimetric analyses. The antimicrobial activity of both PTF and thymol was evaluated against different microorganisms, including S. aureus, E. coli, P. aeruginosa, and C. albicans. The obtained MIC values showed a higher potential for PTF than the monomer thymol-for example, against S. aureus (500 and 31.5 µg·mL-1 for thymol and PTF, respectively). Therefore, the obtained results show that the polymerization of thymol afforded more active biomaterial than the starting monomeric antimicrobial compound (thymol), suggesting that PTF is an important biomaterial.

Micro-Addition of Silver to Copper: One Small Step in Composition, a Change for a Giant Leap in Biocidal Activity.

The use of copper as an antimicrobial agent has a long history and has gained renewed interest in the context of the COVID-19 pandemic. In this study, the authors investigated the antimicrobial properties of an alloy composed of copper with a small percentage of silver (Cu-0.03% wt.Ag). The alloy was tested against various pathogens, including Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, and the H1N1 virus, using contact exposure tests. Results showed that the alloy was capable of inactivating these pathogens in two hours or less, indicating its strong antimicrobial activity. Electrochemical measurements were also performed, revealing that the small addition of silver to copper promoted a higher resistance to corrosion and shifted the formation of copper ions to higher potentials. This shift led to a slow but continuous release of Cu2+ ions, which have high biocidal activity. These findings show that the addition of small amounts of silver to copper can enhance its biocidal properties and improve its effectiveness as an antimicrobial material.

Atlantic Forest's and Caatinga's semiarid soils and their potential as a source for halothermotolerant actinomycetes and proteolytic enzymes.

Actinomycetes are versatile about their metabolism, displaying high capacity to produce bioactive metabolites. Enzymes from actinomycetes represent new opportunities for industrial applications. However, proteases from actinomycetes are poorly described by literature. Thereby, to verify proteolytic potential of actinomycetes, the present study aimed the investigation of bacterial isolates from Caatinga and Atlantic Forest rhizosphere. Fluorescence resonance energy transfer (FRET) peptide libraries were adopted for the evaluations, since they are faster and more qualitative methods, if compared with others described by most reports. A total of 52 microorganisms were inoculated in different culture media (PMB, potato dextrose agar, brain heart infusion agar, Starch Casein Agar and Reasoner's 2A agar), temperatures (12, 20, 30, 37, 45 and 60°C), and saline conditions (0-4 M NaCl), during 7 days. The actinomycetes named as AC 01, 02 and 52 were selected and showed enzymatic abilities under the peptide probes Abz-KLRSSKQ-EDDnp and Abz-KLYSSKQ-EDDnp, achieving enhanced performance at 30 °C. Biochemical parameters were established, showing a predominance of alkaline proteases with activity under saline conditions. Secreted proteases hydrolysed preferentially polar uncharged residues (Y and N) and positively charged groups (R). Phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid inhibited the proteins, a characteristic of serine (AC 01 e 02) and metalloproteases (AC 52). All selected strains belonged to Streptomyces genera. In summary, actinomycete strains with halophilic proteolytic abilities were selected, which improve possibilities for their use in detergent formulations, food processing, waste management and industrial bioconversion. It is important to highlight that this is the first report using FRET libraries for proteolytic screening from Caatinga and Atlantic Forest actinobacteria.

Enzymatic diversity of filamentous fungi isolated from forest soil incremented by sugar cane solid waste.

Fungi are natural degraders of organic matter which can produce enzymes for many industrial and biotechnological applications. In this context, crude enzymatic extracts of fungal isolates were evaluated regarding their hydrolytic and ligninolytic abilities. The fungal strains were isolated from soil samples from Atlantic Rain Forest Park incremented with sugar cane biomass (filter cake), which allowed the selection of efficient lignocellulolytic enzymes. A total of 190 fungi were isolated and evaluated by endocellulase screenings. Thirteen fungi were selected about their hydrolytic and ligninolytic abilities. Among them, three isolates showed xylanolytic activity. Eleven of the isolates were selected by their cellulolytic abilities. Proteolytic enzymes were also detected for three fungi, allowing the classification as metalloprotease and serine protease. The isolates SPZPF3_47 (Mucor sp.), SPZPF1_129 (Byssochlamys nivea) and SPZPF1_141 (Paecilomyces saturatus) were selected for further investigation on their lignin peroxidase abilities. KM, Vmax and kcat apparent for lignin peroxidases were also determined. The strain of Mucor sp. (SPZPF3_47) was highlighted since this fungal genus was not well described about its isolation in the adopted conditions in our study, and showing ligninolytic abilities.

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.

Enantioselective ene-reduction of E-2-cyano-3-(furan-2-yl) acrylamide by marine and terrestrial fungi and absolute configuration of (R)-2-cyano-3-(furan-2-yl) propanamide determined by calculations of electronic circular dichroism (ECD) spectra.

This work reports the green organic chemistry synthesis of E-2-cyano-3(furan-2-yl) acrylamide under microwave radiation (55 W), as well as the use of filamentous marine and terrestrial-derived fungi, in the first ene-reduction of 2-cyano-3-(furan-2-yl) acrylamide to (R)-2-cyano-3-(furan-2-yl)propanamide. The fungal strains screened included Penicillium citrinum CBMAI 1186, Trichoderma sp. CBMAI 932 and Aspergillus sydowii CBMAI 935, and the filamentous terrestrial fungi Aspergillus sp. FPZSP 146 and Aspergillus sp. FPZSP 152. A compound with an uncommon CN-bearing stereogenic center at the α-C position was obtained by enantioselective reactions mediated in the presence of the microorganisms yielding the (R)-2-cyano-3-(furan-2-yl) propanamide 3a. Its isolated yield and e.e. ranged from 86% to 98% and 39% to 99%, respectively. The absolute configuration of the biotransformation products was determined by time-dependent density functional theory (TD-DFT) calculations of electronic circular dichroism (ECD) spectra. Finally, the tautomerization of 2-cyano-3-(furan-2-yl) propanamide 3a to form an achiral ketenimine was observed and investigated in presence of protic solvents.

A Tropical Composting Operation Unit at São Paulo Zoo as a Source of Bacterial Proteolytic Enzymes.

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.

Functional and genetic characterization of hydrocarbon biodegrader and exopolymer-producing clones from a petroleum reservoir metagenomic library.

Microbial degradation of petroleum is a worldwide issue, which causes physico-chemical changes in its compounds, diminishing its commercial value. Biosurfactants are chemically diverse molecules that can be produced by several microorganisms and can enable microbial access to hydrocarbons. In order to investigate both microbial activities, function-driven screening assays for biosurfactant production and hydrocarbon biodegradation were carried out from a metagenomic fosmid library. It was constructed from the total DNA extracted from aerobic and anaerobic enrichments from a Brazilian biodegraded petroleum sample. A sum of 10 clones were selected in order to evaluate their ability to produce exopolymers (EPS) with emulsifying activity, as well as to characterize the gene sequences, harbored by the fosmid clones, through 454 pyrosequencing. Functional analyses confirmed the ability of some clones to produce surfactant compounds. Regarding hydrocarbon as microbial carbon sources, n-alkane (in mixture or not) and naphthalene were preferentially consumed as substrates. Analysis of sequence data set revealed the presence of genes related to xenobiotics biodegradation and carbohydrate metabolism. These data were corroborated by the results of hydrocarbon biodegradation and biosurfactant production detected in the evaluated clones.

Could petroleum biodegradation be a joint achievement of aerobic and anaerobic microrganisms in deep sea reservoirs?

Several studies suggest that petroleum biodegradation can be achieved by either aerobic or anaerobic microorganisms, depending on oxygen input or other electron acceptors and appropriate nutrients. Evidence from in vitro experiments with samples of petroleum formation water and oils from Pampo Field indicate that petroleum biodegradation is more likely to be a joint achievement of both aerobic and anaerobic bacterial consortium, refining our previous observations of aerobic degradation. The aerobic consortium depleted, in decreasing order, hydrocarbons > hopanes > steranes > tricyclic terpanes while the anaerobic consortium depleted hydrocarbons > steranes > hopanes > tricyclic terpanes. The oxygen content of the mixed consortia was measured from time to time revealing alternating periods of microaerobicity (O2 ~0.8 mg.L-1) and of aerobicity (O2~6.0 mg.L-1). In this experiment, the petroleum biodegradation changed from time to time, alternating periods of biodegradation similar to the aerobic process and periods of biodegradation similar to the anaerobic process. The consortia showed preferences for metabolizing hydrocarbons > hopanes > steranes > tricyclic terpanes during a 90-day period, after which this trend changed and steranes were more biodegraded than hopanes. The analysis of aerobic oil degrading microbiota by the 16S rRNA gene clone library detected the presence of Bacillus, Brevibacterium, Mesorhizobium and Achromobacter, and the analysis of the anaerobic oil degrading microbiota using the same technique detected the presence of Bacillus and Acinetobacter (facultative strains). In the mixed consortia Stenotrophomonas, Brevibacterium, Bacillus, Rhizobium, Achromobacter and 5% uncultured bacteria were detected. This is certainly a new contribution to the study of reservoir biodegradation processes, combining two of the more important accepted hypotheses.

Searching for monooxygenases and hydrolases in bacteria from an extreme environment.

Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities.

Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin.

The aim of this study was to characterize and compare the bacterial community structure of two distinct oil samples from a petroleum field in Brazil by using both molecular, based on the construction of 16S rRNA gene libraries, and cultivation methods. Statistical comparisons of libraries based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) data revealed no significant differences between the communities recovered in the non-biodegraded (NBD) and highly biodegraded oils (HBD). BlastN analysis of the 16S rRNA gene sequences representative of distinct ribotypes from both oils showed the presence of nine different bacterial genera in these samples, encompassing members of the genera Arcobacter, Halanaerobium, Marinobacter, Propionibacterium, Streptomyces, Leuconostoc, Acinetobacter, Bacillus and Streptococcus. Enrichments obtained using oil as inoculum and sole carbon source yielded bacterial isolates showing high 16S rRNA gene sequence similarity with Achromobacter xylosoxidans, Bacillus subtilis, Brevibacillus sp., Dietzia sp. and Methylobacterium sp. Comparison between the data obtained using cultivation-independent and enrichment cultures suggests that different selection of community members may occur when using distinct approaches. All the organisms found, except for Leuconostoc sp. and Streptococus sp., have been previously reported in the literature as hydrocarbon degraders and/or associated to oil field environments.