Caatinga, Amazon and Atlantic Forest as natural sources for microbial lignocellulolytic enzymes.

Brazilian biomes are important sources for environmental microorganisms, including efficient metabolic machineries, like actinomycetes. These bacteria are known for their abilities to produce many bioactive compounds, including enzymes with multiple industrial applications. The present work aimed to evaluate lignocellulolytic abilities of actinomycetes isolated from soil and rhizosphere samples collected at Caatinga, Atlantic and Amazon Forest. Laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and cellulase were evaluated for their efficiency. These enzymes have an essential role in lignin decomposition, through oxidation of phenolic and non-phenolic compounds, as well as enzymatic hydrolysis of vegetal biomass. In this sense, a total of 173 actinomycetes were investigated. Eleven (11) of them were selected by their enzymatic performance. The actinomycete AC166 displayed some activity in all analysed scenarios in terms of Lac, MnP and LiP activity, while AC171 was selected as the most promising strain, showing the following activities: 29.7 U.L-1 for Lac; 2.5 U.L-1 for LiP and 23 U.L-1 for MnP. Cellulolytic activities were evaluated at two pH conditions, 4.8 and 7.4, obtaining the following results: 25 U.L-1 and 71 U.L-1, respectively. Thermostability (4, 30 and 60 o C) and salinity concentrations (0 to 4 M) and pH variation (2.0 to 9.0) stabilities of the obtained LiP and Lac enzymatic extracts were also verified. The actinomycete strain AC171 displayed an adaptable response in distinct pH and salt profiles, indicating that bacterial LiP was some halophilic type. Additionally, the strain AC149 produced an alkali and extreme halophilic lignin peroxidase, which are promising profiles for their future application under lignocellulosic biomass at bioethanol biorefineries.

Bypass gastroplasty impacts oral health, salivary inflammatory biomarkers, and microbiota: a controlled study.

OBJECTIVES: Knowledge about the impact of gastroplasty on oral health and salivary biomarkers is limited. The aim was to prospectively evaluate oral health status, salivary inflammatory markers, and microbiota in patients undergoing gastroplasty compared with a control group undergoing a dietary program. MATERIALS AND METHODS: Forty participants with obesity class II/III were included (20 individuals in each sex-matched group; 23-44 years). Dental status, salivary flow, buffering capacity, inflammatory cytokines, and uric acid were assessed. Salivary microbiological analysis (16S-rRNA sequencing) assessed the abundance of genus, species, and alpha diversity. Cluster analysis and mixed-model ANOVA were applied. RESULTS: Oral health status, waist-to-hip ratio, and salivary alpha diversity were associated at baseline. A subtle improvement in food consumption markers was observed, although caries activity increased in both groups, and the gastroplasty group showed worse periodontal status after three months. IFNγ and IL10 levels decreased in the gastroplasty group at 3 months, while a decrease was observed in the control group at 6 months; IL6 decreased in both groups (p < 0.001). Salivary flow and buffering capacity did not change. Significant changes in Prevotella nigrescens and Porphyromonas endodontalis abundance were observed in both groups, while alpha diversity (Sobs, Chao1, Ace, Shannon, and Simpson) increased in the gastroplasty group. CONCLUSIONS: Both interventions changed in different degrees the salivary inflammatory biomarkers and microbiota, but did not improve the periodontal status after 6 months. CLINICAL RELEVANCE: Although the observed discrete improvement in dietary habits, caries activity increased with no clinical improvement in the periodontal status, emphasizing the need of oral health monitoring during obesity treatment.

Degradation of amoxicillin applying photo-Fenton and acid hydrolysis processes with toxicity evaluation via antimicrobial susceptibility tests.

ABSTARCTThe antibiotic amoxicillin (AMX) is a semisynthetic aminopenicillin, classified as an ß-lactam antibiotic. This work aims to evaluate the AMX degradation (190 mg L-1), in aqueous medium, applying photo-Fenton ([TOC]0 = 100 mgC L-1; FH2O2 = 3.27 mmol min-1; [Fe2+] = 0.27 mmol L-1; pH = 3.0; T = 40°C) and acid hydrolysis processes. Along the experiments, samples were withdrawn and analyzed by a total organic carbon (TOC) analyzer and a liquid chromatography system coupled to diode array (HPLC-DAD) and mass spectrometry (HPLC-MS) detectors. The hydrolysis process proved to be less efficient, because AMX removals greater than 80% were observed only after 24 hours of reaction (pH 2). Conversely, the photo-Fenton process removed completely AMX in just 20 minutes, reaching 85% of TOC removal in 2 hours. Finally, the AMX aqueous solutions treated by the studied processes was also evaluated in respect of its toxicity to some microorganisms, applying two antimicrobial susceptibility tests: disk-diffusion and broth microdilution methods. It was observed that the AMX aqueous solutions, pretreated by the photo-Fenton process, for just 7.5 min of reaction time, did not inhibit the microorganisms growth. The obtained results show that the photo-Fenton process was able to degrade AMX, in a relatively short time, and that the generated degradation products did not inhibit the microorganisms growth, when compared to acid hydrolysis process. Thus, it was verified the potential application of the photo-Fenton system as a pretreatment step to conventional biological oxidation processes for the treatment of industrial wastewaters.

Enzymatic potential and biosurfactant production by endophytic fungi from mangrove forest in Southeastern Brazil.

Microbial activity is the main route for cycling mangrove nutrients. In general, microorganisms have abilities to degrade lignocellulosic compounds. Among the biotechnological potential of the microbiota from mangroves, it is noteworthy about endophytic fungi, which can be considered as effective sources of different bioactive compounds. In this sense, thirty (30) endophytic fungi were isolated from mangrove forest sampling Cananeia, SP, Brazil. These microorganisms were analyzed about their enzymatic activities including: lignin peroxidase EC 1.11.1.14, manganese peroxidase EC 1.11.1.13 and laccase EC 1.10.3.2, as well endo-cellulase EC 3.2.1.4 and endo-xylanase EC 3.2.1.8. Besides that, production of bioactive secondary metabolites like biosurfactant and/or bioemulsifier was also investigated. As results, nineteen (19) isolates were selected about their ligninolytic abilities, nine (9) of them about cellulase activity and thirteen (13) showed xylanase abilities. The fungal isolate named as 3(3), characterized as Fusarium sambucinum, showed a prominent lignin peroxidase (42.4 U L-1) and manganese peroxidase (23.6 U L-1) activities. The isolate 63.1, also related to Fusarium sp. genera, was selected about its laccase activity (41.5 U L-1). From all the investigated fungi, the isolate 47(4) Trichoderma camerunense was selected about its cellulolytic and xylanolytic activities, showing 45.23 and 26.09 U mL-1, respectively. The same fungi also showed biosurfactant ability demonstrated by superficial tension decreasing to 38 mN/m. In addition, fifteen (15) fungi exhibited bioemulsifier activity, with E24 values up to 62.8%.

Characterization of novel hydrocarbon-degrading Gordonia paraffinivorans and Gordonia sihwensis strains isolated from composting.

Hydrocarbons are important environmental pollutants, and the isolation and characterization of new microorganisms with the ability to degrade these compounds are important for effective biodegradation. In this work we isolated and characterized several bacterial isolates from compost, a substrate rich in microbial diversity. The isolates were obtained from selective culture medium containing n-hexadecane, aiming to recover alkane-degraders. Six isolates identified as Gordonia by MALDI-TOF and 16S rRNA sequencing had the ability to degrade n-hexadecane in three days. Two isolates were selected for genomic and functional characterization, Gordonia paraffinivorans (MTZ052) and Gordonia sihwensis (MTZ096). The CG-MS results showed distinct n-hexadecane degradation rates for MTZ052 and MTZ096 (86% and 100% respectively). The genome sequence showed that MTZ052 encodes only one alkane degrading gene cluster, the CYP153 system, while MTZ096 harbors both the Alkane Hydroxylase (AH) and the CYP153 systems. qPCR showed that both gene clusters are induced by the presence of n-hexadecane in the growth medium, suggesting that G. paraffinivorans and G. sihwensis use these systems for degradation. Altogether, our results indicate that these Gordonia isolates have a good potential for biotransformation of hydrocarbons.

Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol.

Glycerol is a major byproduct of biodiesel production, and enzymes that oxidize this compound have been long sought after. The recently described alcohol oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here, we describe the comprehensive structural and biochemical characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active toward methanol, ethanol, and 1-propanol ( kcat = 18, 19, and 11 s-1, respectively), but showed very limited activity toward glycerol ( kobs = 0.2 s-1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was determined at a resolution of 2.6 Å. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the observed preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity toward glycerol. Among them, the F101S variant had a high kcat value of 3 s-1, retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation.

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.

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.

Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus.

The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolytic activities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolytic activities, with relevance for industrial applications.

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts.

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

Bioremediation potential of microorganisms derived from petroleum reservoirs.

Bacterial strains and metagenomic clones, both obtained from petroleum reservoirs, were evaluated for petroleum degradation abilities either individually or in pools using seawater microcosms for 21 days. Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) analyses were carried out to evaluate crude oil degradation. The results showed that metagenomic clones 1A and 2B were able to biodegrade n-alkanes (C14 to C33) and isoprenoids (phytane and pristane), with rates ranging from 31% to 47%, respectively. The bacteria Dietzia maris CBMAI 705 and Micrococcus sp. CBMAI 636 showed higher rates reaching 99% after 21 days. The metagenomic clone pool biodegraded these compounds at rates ranging from 11% to 45%. Regarding aromatic compound biodegradation, metagenomic clones 2B and 10A were able to biodegrade up to 94% of phenanthrene and methylphenanthrenes (3-MP, 2-MP, 9-MP and 1-MP) with rates ranging from 55% to 70% after 21 days, while the bacteria Dietzia maris CBMAI 705 and Micrococcus sp. CBMAI 636 were able to biodegrade 63% and up to 99% of phenanthrene, respectively, and methylphenanthrenes (3-MP, 2-MP, 9-MP and 1-MP) with rates ranging from 23% to 99% after 21 days. In this work, isolated strains as well as metagenomic clones were capable of degrading several petroleum compounds, revealing an innovative strategy and a great potential for further biotechnological and bioremediation applications.

New hydrocarbon degradation pathways in the microbial metagenome from Brazilian petroleum reservoirs.

Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs.