Marine Bioprospecting, Biocatalysis and Process Development.

Oceans possess tremendous diversity in microbial life. The enzymatic machinery that marine bacteria present is the result of extensive evolution to assist cell survival under the harsh and continuously changing conditions found in the marine environment. Several bacterial cells and enzymes are already used at an industrial scale, but novel biocatalysts are still needed for sustainable industrial applications, with benefits for both public health and the environment. Metagenomic techniques have enabled the discovery of novel biocatalysts, biosynthetic pathways, and microbial identification without their cultivation. However, a key stage for application of novel biocatalysts is the need for rapid evaluation of the feasibility of the bioprocess. Cultivation of not-yet-cultured bacteria is challenging and requires new methodologies to enable growth of the bacteria present in collected environmental samples, but, once a bacterium is isolated, its enzyme activities are easily measured. High-throughput screening techniques have also been used successfully, and innovative in vitro screening platforms to rapidly identify relevant enzymatic activities continue to improve. Small-scale approaches and process integration could improve the study and development of new bioprocesses to produce commercially interesting products. In this work, the latest studies related to (i) the growth of marine bacteria under laboratorial conditions, (ii) screening techniques for bioprospecting, and (iii) bioprocess development using microreactors and miniaturized systems are reviewed and discussed.

Cultivating marine bacteria under laboratory conditions: Overcoming the "unculturable" dogma.

Underexplored seawater environments may contain biological resources with potential for new biotechnological applications. Metagenomic techniques revolutionized the study of bacterial communities but culture dependent methods will still be important to help the biodiscovery of new products and enzymes from marine bacteria. In this context, we promoted the growth of bacteria from a marine rock pond by culture dependent techniques and compared the results with culture independent methods. The total number of bacteria and diversity were studied in different agar plate media during 6 weeks. Agar plate counting was of the same order of magnitude of direct microscopy counts. The highest efficiency of cultivation was 45% attained in marine agar medium. Molecular analysis revealed 10 different phyla of which only four were isolated by the culture dependent method. On the other hand, four taxonomic orders were detected by cultivation but not by the molecular technique. These include bacteria from the phyla Bacillota and Actinomycetota. Our study shows that it is possible to grow more than the traditionally considered 1% of bacteria from a seawater sample using standard agar plate techniques and laboratorial conditions. The results also demonstrate the importance of culture methods to grow bacteria not detected by molecular approaches for future biotechnological applications.

Process Development for Benzyl Alcohol Production by Whole-Cell Biocatalysis in Stirred and Packed Bed Reactors.

The ocean is an excellent source for new biocatalysts due to the tremendous genetic diversity of marine microorganisms, and it may contribute to the development of sustainable industrial processes. A marine bacterium was isolated and selected for the conversion of benzaldehyde to benzyl alcohol, which is an important chemical employed as a precursor for producing esters for cosmetics and other industries. Enzymatic production routes are of interest for sustainable processes. To overcome benzaldehyde low water solubility, DMSO was used as a biocompatible cosolvent up to a concentration of 10% (v/v). A two-phase system with n-hexane, n-heptane, or n-hexadecane as organic phase allowed at least a 44% higher relative conversion of benzaldehyde than the aqueous system, and allowed higher initial substrate concentrations. Cell performance decreased with increasing product concentration but immobilization of cells in alginate improved four-fold the robustness of the biocatalyst: free and immobilized cells were inhibited at concentrations of benzyl alcohol of 5 and 20 mM, respectively. Scaling up to a 100 mL stirred reactor, using a fed-batch approach, enabled a 1.5-fold increase in benzyl alcohol productivity when compared with batch mode. However, product accumulation in the reactor hindered the conversion. The use of a continuous flow reactor packed with immobilized cells enabled a 9.5-fold increase in productivity when compared with the fed-batch stirred reactor system.

Determining transaminase activity in bacterial libraries by time-lapse imaging.

Transaminase activity was determined by time-lapse imaging using a colourimetric reaction and image analysis. A correlation between the benzaldehyde conversion and relative luminance was determined, allowing the identification of the most promising biocatalysts, the determination of kinetic parameters, and the assessment of the effect of the substrate concentration on activity.

Phenotypic Adaptations Help Rhodococcus erythropolis Cells during the Degradation of Paraffin Wax.

During crude oil extraction, the reduction in temperature and pressure results in the precipitation of paraffin wax that contains 20-40 carbon chain hydrocarbons. The paraffin wax may accumulate inside production tubes, pipelines, and processing facilities, and also in tankers during petroleum transportation. There are few bacterial strains that are able to degrade solid substrates. In the present study, the biodegradation of paraffin is evaluated using Rhodococcus erythropolis cells. This bacterium is able to grow using paraffin wax from an oil refinery plant as the sole carbon source. The cells grow as a thick biofilm over the solid substrate, make scale-like structures that increase the area of the initially smooth surface of paraffin, produce biosurfactants, and become more negatively charged than ethanol- or glucose-grown cells. When paraffin wax is supplied as microparticles, to increase the cell-substrate contact area and to simulate paraffin precipitation, the cells also adjust the composition of the fatty acids of the phospholipids of the cellular membrane to decrease its fluidity and paraffin biodegradation increases considerably. The study suggests that the phenotypic adaptation of R. erythropolis cells may be used to degrade paraffin wax under real conditions.

Clinical, immunological and microbial gingival profile of juvenile systemic lupus erythematosus patients.

Periodontal disease has been associated with rheumatic diseases; however, few studies have evaluated the association with systemic lupus erythematosus (SLE), and its impact on the local inflammatory and microbial profiles. Therefore, this study evaluated the levels of several cytokines in gingival crevicular fluid (GCF) and serum from juvenile SLE (jSLE) patients with gingival inflammation, compared with controls. In addition, we assessed their subgingival microbial profile. Thirty jSLE patients and 29 systemically healthy individuals were recruited. Participants were rheumatologically and periodontally examined, and GCF, serum and intrasulcular biofilm were collected. Cytokines were analysed by bead-based multiplex assays and the bacterial profile by checkerboard DNA-DNA hybridization. jSLE patients presented higher percentages of dental plaque and bleeding than controls, as well as increased mean probing depth and attachment loss. After adjustment for multiple comparisons, GCF levels of interleukin (IL)-1ß, IL-8, granulocyte colony-stimulating factor (G-CSF), interferon-γ and monocyte chemoattractant protein-1 were significantly higher, whereas the levels of granulocyte-macrophage colony-stimulating factor were significantly lower in jSLE patients. In serum, G-CSF levels tended to be higher in jSLE patients (adjusted p-value = 0.06). Intrasulcular counts of Aggregatibacter actinomycetemcomitans were significantly higher in jSLE patients as compared with controls. We conclude that patients with jSLE present a worse periodontal condition associated with altered levels of pro-inflammatory cytokines in GCF and increased counts of A. actinomycetemcomitans in the intrasulcular biofilm.

Cultivation-based strategies to find efficient marine biocatalysts.

Marine bacteria have evolved to survive in the marine environment by using unique physiological, biochemical and metabolic features and the ability to produce enzymes and compounds which may have commercial value. The Azores archipelago presents several ecosystems with strong volcanic activity where bacteria thrive under e.g. high temperatures. In this study, samples collected in the island of São Miguel were screened for biocatalysts possessing e.g. lipase, esterase, amylase, and inulinase activities. After isolation of several hundred bacterial strains, high throughput screening methods allowed the fast identification of biocatalysts. The first cultivation tests were performed on 24-wells microtiter plates with online oxygen monitoring and bacteria able to grow within 24 h were selected for further process development. Bacteria able to produce the desired enzymes were selected for the first round of tests. Four Bacillus strains presented high inulinase activity. The next step in process development was the determination of key parameters for enzyme activity such as temperature, pH, salinity and substrate concentration. The highest inulinase activity, 2.2 gsugars /gprotein h, was attained when the supernatant of a culture of a Bacillus subtilis strain was used in a magnetically stirred bioreactor. This study demonstrates how bacterial strains from marine environments may be used successfully in biotechnological processes.

Rhodococcus erythropolis cells adapt their fatty acid composition during biofilm formation on metallic and non-metallic surfaces.

Several parameters are involved in bacterial adhesion and biofilm formation including surface type, medium composition and cellular surface hydrophobicty. When the cells are placed inside tubes, parameters such as oxygen availability should also influence cell adhesion. To understand which cellular lipids are involved in the molecular events of biofilm formation in Rhodococcus erythropolis, cell adhesion was promoted on different metallic and non-metallic surfaces immersed in culture media. These cells were able to modulate the fatty acid composition of the cell membrane in response to both the surface to which they adhered and the growth medium used. To assess the response of the cells to both surfaces and operational conditions, biofilms were also promoted inside a reactor built with five different types of tubes and with medium recirculation. The biofilm biomass could be directly related not to the hydrophobicity of the tubes used but to the oxygen permeability of the tubes. Besides this, cell age influenced the adhesion of the R. erythropolis cells to the tubes. Principal component analysis showed that the lipid composition of the cells could separate cells attached to metallic from those on non-metallic surfaces in the plane formed by PC1 and PC2, and influence biofilm biomass.

Call recognition and individual identification of fish vocalizations based on automatic speech recognition: An example with the Lusitanian toadfish.

The study of acoustic communication in animals often requires not only the recognition of species specific acoustic signals but also the identification of individual subjects, all in a complex acoustic background. Moreover, when very long recordings are to be analyzed, automatic recognition and identification processes are invaluable tools to extract the relevant biological information. A pattern recognition methodology based on hidden Markov models is presented inspired by successful results obtained in the most widely known and complex acoustical communication signal: human speech. This methodology was applied here for the first time to the detection and recognition of fish acoustic signals, specifically in a stream of round-the-clock recordings of Lusitanian toadfish (Halobatrachus didactylus) in their natural estuarine habitat. The results show that this methodology is able not only to detect the mating sounds (boatwhistles) but also to identify individual male toadfish, reaching an identification rate of ca. 95%. Moreover this method also proved to be a powerful tool to assess signal durations in large data sets. However, the system failed in recognizing other sound types.

C. albicans, C. parapsilosis and C. tropicalis invasive infections in the PICU: clinical features, prognosis and mortality.

Candida albicans remains the most common agent associated with invasive Candida infection (ICI), but with increasing number of non-albicans species. An epidemiological, observational study exploring host criteria, clinical characteristics and mortality of ICI was performed in 24 pediatric intensive care units (PICU) in Spain. Patients were analyzed in global and distributed by infecting species (for groups with ≥ 15 patients). A total of 125 ICI were included: 47 by C. albicans, 37 by C. parapsilosis, 19 by C. tropicalis, 4 C. glabrata, and 18 others. Up to 66% of ICI by C. albicans and 75.7% by C. parapsilosis occurred in children ≤ 24 months, while the percentage of children >60 months was higher in ICI by C. tropicalis. Bloodstream infection was most common among C. tropicalis (78.9%) or C. parapsilosis (83.8%) ICI, but urinary infections were almost as common as bloodstream infections among C. albicans ICI (31.9% and 38.3%, respectively). Fever refractory to antimicrobials was the most frequent host criterion (46.4% patients), but with equal frequency than prolonged neutropenia in C. tropicalis ICI. Thrombopenia was more frequent (p<0.05) in C. parapsilosis (60.7%) or C. tropicalis (66.7%) ICI than in C. albicans ICI (26.5%). Uremia was more frequent (p<0.05) in C. albicans (78.3%) or C. tropicalis (73.3%) than in C. parapsilosis ICI (40.7%). Multiple organ failure and heart insufficiency was higher in C. tropicalis ICI. Short duration (≤ 7 days) of PICU stay was more frequent in C. albicans ICI. Mortality rates were: 8.5% (C. albicans ICI), 13.5% (C. parapsilosis ICI) and 23.3% (C. tropicalis ICI). ICI by different Candida species showed different clinical profiles and mortality, making essential identification at species level.

Digestion in adult females of the leaf-footed bug Leptoglossus zonatus (Hemiptera: Coreidae) with emphasis on the glycoside hydrolases α-amylase, α-galactosidase, and α-glucosidase.

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α-glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane-bound isoforms, being more abundant as a membrane-bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion-exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α-amylases and α-galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α-amylases acting on maize seed starch granules.

Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus.

BACKGROUND: Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control. METHODS: SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes. RESULTS: The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1' was translocated to the putative position P4'-P5'. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three ß-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus. CONCLUSIONS: For the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule.

Metabolic signatures of triatomine vectors of Trypanosoma cruzi unveiled by metabolomics.

Chagas disease is a trypanosomiasis whose causative agent is the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by hematophagous insects known as triatomines and affects a large proportion of South America. The digestive tract of the insect vectors in which T. cruzi develops constitutes a dynamic environment that affects the development of the parasite. Thus, we set out to investigate the chemical composition of the triatomine intestinal tract through a metabolomics approach. We performed Direct Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry on fecal samples of three triatomine species (Rhodnius prolixus, Triatoma infestans, Panstrongylus megistus) fed with rabbit blood. We then identified groups of metabolites whose frequencies were either uniform in all species or enriched in each of them. By querying the Human Metabolome Database, we obtained putative identities of the metabolites of interest. We found that a core group of metabolites with uniform frequencies in all species represented approximately 80% of the molecules detected, whereas the other 20% varied among triatomine species. The uniform core was composed of metabolites of various categories, including fatty acids, steroids, glycerolipids, nucleotides, sugars, and others. Nevertheless, the metabolic fingerprint of triatomine feces differs depending on the species considered. The variable core was mainly composed of prenol lipids, amino acids, glycerolipids, steroids, phenols, fatty acids and derivatives, benzoic acid and derivatives, flavonoids, glycerophospholipids, benzopyrans, and quinolines. Triatomine feces constitute a rich and varied chemical medium whose constituents are likely to affect T. cruzi development and infectivity. The complexity of the fecal metabolome of triatomines suggests that it may affect triatomine vector competence for specific T. cruzi strains. Knowledge of the chemical environment of T. cruzi in its invertebrate host is likely to generate new ways to understand the factors influencing parasite proliferation as well as methods to control Chagas disease.

Varicela hemorrágica asociada a síndrome de distrés respiratorio agudo / Hemorrhagic chickenpox associated to acute respiratory stress syndrome

Niño de 5 años de edad que ingresa en la UCI por insuficiencia respiratoria severa con el antecedente de una varicela clínica de 10 días de evolución. El paciente presenta un mal estado general, palidez cutánea cérea generalizada, mala perfusión periférica y piel fría. Se observaron abundantes lesiones cutáneas vesiculosas hemorrágicas generalizadas. En la radiografía de tórax se observó condensación algodonosa del parénquima bilateral compatible con síndrome de distrés respiratorio agudo (SDRA) y probable neumotoráx derecho basal con derrame pleura. Los resultados microbiológicos fueron negativos salvo el aislamiento de virus varicela-zoster en el exudado de las lesiones cutáneas. Asimismo, la técnica de amplificación genómica (PCR) fue positiva para varicela zóster en el aspirado traqueal y el derrame pleural. Al tercer día de su ingreso el paciente presentó un episodio brusco de hipotensión arterial, con sangrado activo por fosas nasales y boca y fallecimiento (AU)

Children of 5 years old who is admitted to the ICU for severe respiratory failure with a history of clinical varicella with 10 days of evolution. The patient presented with malaise, generalized waxy pallor, poor peripheral perfusion and cold skin. There were plenty of vesicular skin lesions generalized bleeding. The chest radiograph showed bilateral condensation cottony parenchyma compatible with acute respiratory distress syndrome (ARDS) and right pneumothorax with basal pleural effusion. The microbiological results were negative except the isolation of varicella-zoster virus in the exudate of the skin lesions. Like wise, the genomic amplification technique (PCR) was positive for varicella-zoster virus in tracheal aspirate and pleural effusion. On the third day of admission the patient presented a sudden episode of hypotension, with active bleeding from nose and mouth and died (AU)