BIOREFINERY FOR BIOPOLYMERS:NEW TOOLS FOR BIOMATERIALS PRODUCTION, DEGRADATION AND SUSTAINABLE FUNCTIONALIZATION

Biopolymers are attractive “green” alternatives to conventional petroleum-based plastics, however, their sustainable exploitation is hampered by the high production costs. In this PhD thesis, an Escherichia coli recombinant system (LipoA) was constructed to allow production of Polyhydroxyalkanoates (PHAs). The system was engineered with a newly isolated PHAs biosynthetic operon from Bacillus cereus 6E/2 and tested for PHAs production on different carbon sources. Results highlighted the LipoA peculiar specificity to drive the incorporation of 3-hydroxyhexanoate monomers (>40%), whatever was the supplied fatty acid. To increase polymer production, media optimization and system engineering were applied. In this frame, two new PHA producing systems were developed considering i) the expression levels of the recombinant PHA bio-synthetic proteins (LipoB) and ii) the "host metabolic background" (LipoC) yielding to a 6-fold increment of mcl-PHA yields. Polymers were characterized revealing a low grade of crystallinity and hydrophobic features. To enhance biopolymer properties and expand fields of applicability, PHAs were enzymatically functionalized. Commercial lipase B from Candida antarctica (CaLB) was able to catalyse coupling of PHA with dimethyl itaconate (DMI) as well as with polyethylene glycol (PEG). The obtained functional hydrophilic biopolymers open new perspectives for application of PHAs in the biomedical field thanks to the possibility of easy coupling of bioactive compounds on the lateral C=C of DMI and to the enhanced hydrophilicity conferred by the PEG moieties. The enzymatic strategy was also applied for sustainable synthesis of oligoesters. Catalytic potential of immobilized Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) was investigated. Three different carriers, linked to the enzyme using a novel nontoxic His-tag method based on chelated Fe(III) ions, were tested. Selectivity chain (diols-diesters) and recyclability studies in solvent-free environment were conducted. Results not only revealed a peculiar substrates specificity but also a retention of activity >94 % over 24 h reaction cycle claiming the potentiality of new immobilization strategy. In addition, degrading capabilities of this enzyme against aromatic (PET) and aliphatic biopolyesters (PBS, PHBV, PLA) were investigated. Two glycosylation sites knock out mutants, recombinantly expressed in Pichia pastoris, were tested in comparison to wild-type (wt) enzyme. Data claimed that rThc_Cut1 and its mutants hydrolyse aromatic and aliphatic polyester powders at different rates. The best performances were observed against PBS with concentration of released products 10-fold higher than PHBV and PLA. It is worth of note that one mutant was found to be significantly more active on both powder and PBS films than the wt. These results together with the high activity of variants of rThc_Cut1 on PET provide a significant contribution toward enzymatic degradation of polyesters. To make PHA production environmental sustainable and economically competitive, the use of inexpensive substrates was also investigated: waste frying oils (WFOs) were tested as substrates. The commercial value of WFO is depending on several factors and the free fatty acids (FFAs) content strongly decrease the price since it strongly affects the yield of the biodiesel production process. Microbial PHA production alternative to acid pre-treatment of FFAs-rich WFO was explored. The introduction of an upstream microbial fermentation step of ad hoc systems achieves the 2-fold purpose of reduction of the FFAs content of the waste and of producing added-value products. Lab-scale results proved the exploitability of the proposed bioprocess both with recombinant and native PHA producing cell factories. In the case of native microorganism, the effect of extracellular lipase in biopolymers production and FFAs reduction was also investigated. Moreover, to re-introduce into the production flow a by-product of biodiesel production, glycerol was tested as C-source for fermentation. This substrate was applied for boosting PHAs production in above-mentioned bioprocess as well as main C-source for properly designed recombinant E. coli strains (Omni strains). The investigation of these systems on media supporting production of different PHA co-polymers laid the foundation for the selection and enhancement of enzymes and metabolic background contributing to biopolymers production

CO2-Induced Transcriptional Reorganization: Molecular Basis of Capnophillic Lactic Fermentation in Thermotoga neapolitana

Capnophilic lactic fermentation (CLF) is a novel anaplerotic pathway able to convert sugars to lactic acid (LA) and hydrogen using CO2 as carbon enhancer in the hyperthermophilic bacterium Thermotoga neapolitana. In order to give further insights into CLF metabolic networks, we investigated the transcriptional modification induced by CO2 using a RNA-seq approach. Transcriptomic analysis revealed 1601 differentially expressed genes (DEGs) in an enriched CO2 atmosphere over a total of 1938 genes of the T. neapolitana genome. Transcription of PFOR and LDH genes belonging to the CLF pathway was up-regulated by CO2 together with 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydratase (EDD) of the Entner–Doudoroff (ED) pathway. The transcriptomic study also revealed up-regulation of genes coding for the flavin-based enzymes NADH-dependent reduced ferredoxin:NADP oxidoreductase (NFN) and NAD-ferredoxin oxidoreductase (RNF) that control supply of reduced ferredoxin and NADH and allow energy conservation-based sodium translocation through the cell membrane. These results support the hypothesis that CO2 induces rearrangement of the central carbon metabolism together with activation of mechanisms that increase availability of the reducing equivalents that are necessary to sustain CLF. In this view, this study reports a first rationale of the molecular basis of CLF in T. neapolitana and provides a list of target genes for the biotechnological implementation of this process

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

Background: Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals. Results: We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa), which specifically binds to human fibronectin (Fn), an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin. Conclusion: Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins. Isolation of LM3-CC1 strain was possible for the presence of expressed enoA2 gene in the L. plantarum genome, giving the possibility, for the first time to our knowledge, to quantitatively compare adhesion of wild type and mutant strain, and to assess doubtless the role of L. plantarum Eno A1 as a fibronectin binding protein. © 2009 Castaldo et al; licensee BioMed Central Ltd

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein.

BACKGROUND: Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals. RESULTS: We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa), which specifically binds to human fibronectin (Fn), an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin. CONCLUSION: Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins. Isolation of LM3-CC1 strain was possible for the presence of expressed enoA2 gene in the L. plantarum genome, giving the possibility, for the first time to our knowledge, to quantitatively compare adhesion of wild type and mutant strain, and to assess doubtless the role of L. plantarum Eno A1 as a fibronectin binding protein

Effect of Cultivation Parameters on Fermentation and Hydrogen Production in the Phylum

The phylum is composed of a single class (), 4 orders (), 5 families (), and 13 genera. They have been isolated from extremely hot environments whose characteristics are reflected in the metabolic and phenotypic properties of the species. The metabolic versatility of members leads to a pool of high value-added products with application potentials in many industry fields. The low risk of contamination associated with their extreme culture conditions has made most species of the phylum attractive candidates in biotechnological processes. Almost all members of the phylum, especially those in the order , can produce bio-hydrogen from a variety of simple and complex sugars with yields close to the theoretical Thauer limit of 4 mol H/mol consumed glucose. Acetate, lactate, and L-alanine are the major organic end products. Thermotagae fermentation processes are influenced by various factors, such as hydrogen partial pressure, agitation, gas sparging, culture/headspace ratio, inoculum, pH, temperature, nitrogen sources, sulfur sources, inorganic compounds, metal ions, etc. Optimization of these parameters will help to fully unleash the biotechnological potentials of and promote their applications in industry. This article gives an overview of how these operational parameters could impact fermentation in terms of sugar consumption, hydrogen yields, and organic acids production

Pelvic Health Assessment in Adult Females Following Pediatric Appendicitis: A Monocentric Retrospective Case—Control Study

open10noAbstract: Background: The anatomical location of the appendix in females determines its close contact with the internal genitalia, involving the latter in case of acute appendicitis (AA). The aim of this study was to evaluate the incidence of pelvic health impairment in adult women who underwent appendicectomy during childhood. Materials and Methods: A retrospective observational study was conducted including all female patients who underwent appendicectomy for acute appendicitis at our Center between January 1985 and December 1995. The patients were divided into two groups, i.e., complicated AA (Group A) and not complicated AA (Group B), and were asked to respond to a questionnaire investigating their general health status, fertility impairment, ectopic pregnancies, miscarriages, endometriosis, and chronic pelvic pain. The same questionnaire was administered to female volunteers with past medical history (PMH) negative for AA. The data were compared using chi-square test and Fisher exact test (a p value < 0.05 was considered for statistical significance). Results: In total, 75 patients operated for AA during childhood (22 in Group A and 53 in group B) and 44 female volunteers with PMH negative for AA (group C) were enrolled in the study. Seventeen patients (77.3%) in group A, 40 (75.4%) in group B, and 29 (65.9%) in group C (p > 0.05) had pregnancies. The number of miscarriages among women who became pregnant in their life was 5 in group A, 13 in group B, and 12 in group C (p > 0.05). Chronic pelvic pain was reported by 7 out of 22 (31.8%) patients in group A, 7 out of 53 (13.2%) in group B, and 5 out of 44 (11.4%) in group C (A vs. C: p = 0.04, OR = 3.64; A vs. B: p = 0.06 and B vs. C: p = 0.52). Conclusions: In our series, AA, complicated or not, neither determined repercussions on fertility, risk of miscarriages, and ectopic pregnancies nor increased the risk of developing endometriosis. However, women who experienced complicated AA showed a higher prevalence of chronic pelvic pain onset in adulthood compared to healthy women.openParente, Giovanni; Di Mitri, Marco; D’Antonio, Simone; Cravano, Sara; Thomas, Eduje; Vastano, Marzia; Lunca, Robert; Gargano, Tommaso; Libri, Michele; Lima, MarioParente, Giovanni; Di Mitri, Marco; D’Antonio, Simone; Cravano, Sara; Thomas, Eduje; Vastano, Marzia; Lunca, Robert; Gargano, Tommaso; Libri, Michele; Lima, Mari

Characterization of alginate from sargassum duplicatum and the antioxidant effect of alginate-okra fruit extracts combination for wound healing on diabetic mice

Diabetes mellitus is a group of metabolic disorders characterized by high blood-glucose levels over a prolonged period that causes complications when an open wound is present. Alginate is an antioxidant and a good absorbent of exudates. Okra fruit contains flavonoids that can act as antioxidants. The antioxidant properties of extracts combination reduce blood-glucose levels significantly to accelerate the activities of wound-healing processes on diabetic mice. Alginate was characterized by Size Exclusion Chromatography-Multiple Angle Laser Light Scattering (SEC-MALLS), thermal stability and Proton Nuclear Magnetic Resonance (1H-NMR). The evaluation of wound healing on 36 male mice were divided into 12 groups including normal control (NC), diabetics control (DC), alginate (DA) and alginate-okra (DAO) groups in three different times by histopathology test on skin tissue. The results of SEC-MALLS analysis showed that alginate as single and homogeneous polysaccharide. The 1H-NMR spectrum showed that the mannuronate/guluronate ratio of the used alginate was 0.91. Alginate, okra fruit extract and their combination were classified as moderate and strong antioxidants. The numbers of fibrocytes, fibroblasts, collagen densities had significantly increased from three to seven days. In contrast, wound width, neutrophil, macrophages had significantly decreased at 14 days. The administration of extracts combination increased the re-epithelization of the wound area and wound-healing process on diabetic mice

Safer bio-based solvents to replace toluene and tetrahydrofuran for the biocatalyzed synthesis of polyesters

With increased awareness of environmental issues caused by traditional petrochemical processes, both academia and industry are making enormous efforts towards the development of sustainable practices using renewable biomass as a feedstock. In this work, the biocatalyzed synthesis of polyesters derived from renewable monomers was performed in safer, bio-derivable organic solvents. Candida antarctica lipase B (CaLB), an enzyme belonging to the Ser-hydrolase family (adsorbed on methacrylic resin, also known as Novozym 435) was tested for its performance in the synthesis of adipate- and furandicarboxylate-based polyesters. In addition, the traditional solvents toluene and tetrahydrofuran were compared with a series of green solvents, 2,2,5,5-tetramethyloxolane, 2-methyltetrahydrofuran, 2,5-dimethyltetrahydrofuran and pinacolone for the enzymatic polymerizations. We can conclude that the monomer conversions and molecular masses of the obtained polyesters in all the tested alternative solvents were suitable, and in some cases superior, with CaLB immobilized via physisorption on acrylic resin being the optimal biocatalyst for all reactions. Strikingly, it was found that for the majority of the new solvents, lower reaction temperatures gave comparable monomer conversions and polymers with similar molecular weights whilst pinacolone yielded better polymers with Mn > 2000 Da and conversions of over 80%

Ask Doctor Smartphone! An App to Help Physicians Manage Foreign Body Ingestions in Children

Abstract: Background: Foreign body ingestion (FBI) represents the most common cause of emergent gastrointestinal endoscopy in children. FBI’s management can be quite challenging for physicians because of the variability of the clinical presentation, and the decision tree becomes even more intricate because of patient-specific variables that must be considered in the pediatric age range (e.g., age of patients and neuropsychiatric disorders) in addition to the mere characteristics of the foreign body. We present an application for smartphones designed for pediatricians and pediatric surgeons based on the latest guidelines from the official pediatric societies. The app aims to help physicians manage FBI quickly and properly in children. Materials and methods: The latest pediatric FBI management guidelines were reviewed and summarized. The flow chart we obtained guided the development of a smartphone application. A questionnaire was administered to all pediatric surgeon trainees at our institute to test the feasibility and helpfulness of the application. Results: An app for smartphones was obtained and shared for free on the Google Play Store and Apple Store. The app guides the physician step by step in the diagnostic process, analyzing all patient- and foreign body-specific characteristics. The app consultation ends with a suggestion of the most proper decision to make in terms of further radiological investigations and the indication and timing of endoscopy. A questionnaire administered to trainees proved the app to be useful and easy to use. Conclusion: We developed an app able to help pediatricians and pediatric surgeons manage FBI in children, providing standardized and updated recommendations in a smart and easily available way

Sustainable Galactarate-Based Polymers : Multi-Enzymatic Production of Pectin-Derived Polyesters

Large amounts of agricultural wastes are rich in pectins that, in many cases, disrupt the processing of food residues due to gelation. Despite pectins being a promising sustainable feedstock for bio-based chemical production, the current pathways to produce platform molecules from this polysaccharide are hazardous and entail the use of strong acids. The present work describes a sequence of biocatalyzed reactions that involves 1) the extraction of pectin from sugar beet pulp and enzymatic recovery of galacturonic acid (GalA), followed by 2) the enzymatic oxidation of the GalA aldehyde and the recovery of galactaric acid (GA), and 3) the biocatalyzed polycondensation of GA to obtain fully bio-based polyesters carrying lateral hydroxy functionalities. The acid-free pectin extraction is optimized using enzymes and microwave technology. The conditions for enzymatic oxidation of GalA allow the separation of the GA produced by a simple centrifugation step that leads to the enzyme-catalyzed polycondensation reactions