Laccase-assisted bio-grafting of polyhydroxy-alkanoates (PHAs) onto the ethyl cellulose (EC) backbone

In the present study, a novel enzyme-based methodology for grafting Polyhydroxyalkanoates (PHAs) onto the ethyl cellulose (EC) as a backbone polymer was developed. Laccase assisted copolymerization was carried out under mild and eco-friendly reaction conditions. The resulting homogeneous composite membranes were characterized by Fourier-transformed infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Atomic Force Microscopy (AFM). The FTIR spectra of pure PHAs and PHAs containing graft composites (PHAs-g-EC) showed their strong characteristic bands at 1721 cm1, 1651 cm-1 and 1603 cm-1 respectively. Other accompanying bands in the range of 900-1300 cm-1 correspond to C=O vibration and C-O-C bond stretching, which could be contributed from PHAs and EC, respectively. The high intensity of the 3358 cm-1 band in the graft composite may have corresponded to the degradation of the carboxylic group from PHAs and also showed an increase of hydrogen-bonded groups at that distinct band region. The morphology was examined by SEM, which showed the well dispersed PHAs crystals in the backbone polymer of EC. XRD pattern for PHAs showed distinct peaks at 2-Theta values of 28o, 32o, 34o, 39o, 46o, 57o, 64o, 78o and 84o that represent the crystalline nature of PHAs. In comparison with those of neat PHAs, the degree of crystallinity for PHAs-g-EC decreased and this reduction is mainly because of the new cross-linking of PHAs within the EC backbone that changes the morphology and destroys the crystallites. Improved mechanical properties were observed for the PHAs-g-EC as compared to the individual components due to the impregnation of EC as reinforcement into the PHAs matrix. Improved mechanical strength enhanced thermal properties, along with low crystallinity of the present PHAs-g-EC suggesting its potential for various industrial and bio-medical applications.

Desenvolvimento de estratégias para aumentar a produtividade e controlar as propriedades de biopolímeros (PHAs) produzidas por culturas mistas

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestrado em Engenharia Química e Bioquímica

Rhamnolipids and PHAs: Recent reports on Pseudomonas-derived molecules of increasing industrial interest

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo da produção de metabólitos por Pseudomonas aeruginosa: Ramnolipídios e Polihidroxialcanoatos (PHAs)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polihidroxialcanoatos (PHAs): bioprospecção de micro-organismos e produção a partir de glicerol

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

The use of an acetoacetyl-CoA synthase in place of a -ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C-4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing -ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the -ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2x10(6)Da. These results are a major step forward in engineering a high biomass C-4 grass for the commercial production of PHB.

Life cycle assessments of biodegradable, commercial biopolymers - A critical review

Biopolymers are generally considered an eco-friendly alternative to petrochemical polymers due to the renewable feedstock used to produce them and their biodegradability. However, the farming practices used to grow these feedstocks often carry significant environmental burdens, and the production energy can be higher than for petrochemical polymers. Life cycle assessments (LCAs) are available in the literature, which make comparisons between biopolymers and various petrochemical polymers, however the results can be very disparate. This review has therefore been undertaken, focusing on three biodegradable biopolymers, poly(lactic acid) (PLA), poly(hydroxyalkanoates) (PHAs), and starch-based polymers, in an attempt to determine the environmental impact of each in comparison to petrochemical polymers. Reasons are explored for the discrepancies between these published LCAs. The majority of studies focused only on the consumption of non-renewable energy and global warming potential and often found these biopolymers to be superior to petrochemically derived polymers. In contrast, studies which considered other environmental impact categories as well as those which were regional or product specific often found that this conclusion could not be drawn. Despite some unfavorable results for these biopolymers, the immature nature of these technologies needs to be taken into account as future optimization and improvements in process efficiencies are expected. © 2013 Elsevier B.V. All rights reserved.

Study of Methane Oxidative Coupling over Ti-La-Li Mixed Oxides

The composition and structures of Li-Ti-La mixed oxides as well as their catalytic activity for methane oxidative coupling have been studied by means of XRD XPS, IR, SEM and so on. The results indicate that by changing x value in Li-La1-xTixO2 oxides phas

Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions.

Investigation of polyhydroxyalkanoate production by an activated sludge microbial consortium treating artificial dairy wastewater

Petrochemical plastics/polymers are a common feature of day to day living as they occur in packaging, furniture, mobile phones, computers, construction equipment etc. However, these materials are produced from non-renewable materials and are resistant to microbial degradation in the environment. Considerable research has therefore been carried out into the production of sustainable, biodegradable polymers, amenable to microbial catabolism to CO2 and H2O. A key group of microbial polyesters, widely considered as optimal replacement polymers, are the Polyhydroxyalkaonates (PHAs). Primary research in this area has focused on using recombinant pure cultures to optimise PHA yields, however, despite considerable success, the high costs of pure culture fermentation have thus far hindered the commercial viability of PHAs thus produced. In more recent years work has begun to focus on mixed cultures for the optimisation of PHA production, with waste incorporations offering optimal production cost reductions. The scale of dairy processing in Ireland, and the high organic load wastewaters generated, represent an excellent potential substrate for bioconversion to PHAs in a mixed culture system. The current study sought to investigate the potential for such bioconversion in a laboratory scale biological system and to establish key operational and microbial characteristics of same. Two sequencing batch reactors were set up and operated along the lines of an enhanced biological phosphate removal (EBPR) system, which has PHA accumulation as a key step within repeated rounds of anaerobic/aerobic cycling. Influents to the reactors varied only in the carbon sources provided. Reactor 1 received artificial wastewater with acetate alone, which is known to be readily converted to PHA in the anaerobic step of EBPR. Reactor 2 wastewater influent contained acetate and skim milk to imitate a dairy processing effluent. Chemical monitoring of nutrient remediation within the reactors as continuously applied and EBPR consistent performances observed. Qualitative analysis of the sludge was carried out using fluorescence microscopy with Nile Blue A lipophillic stain and PHA production was confirmed in both reactors. Quantitative analysis via HPLC detection of crotonic acid derivatives revealed the fluorescence to be short chain length Polyhydroxybutyrate, with biomass dry weight accumulations of 11% and 13% being observed in reactors 1 and 2, respectively. Gas Chromatography-Mass Spectrometry for medium chain length methyl ester derivatives revealed the presence of hydroxyoctanoic, -decanoic and -dodecanoic acids in reactor 1. Similar analyses in reactor 2 revealed monomers of 3-hydroxydodecenoic and 3-hydroxytetradecanoic acids. Investigation of the microbial ecology of both reactors as conducted in an attempt to identify key species potentially contributing to reactor performance. Culture dependent investigations indicated that quite different communities were present in both reactors. Reactor 1 isolates demonstrated the following species distributions Pseudomonas (82%), Delftia acidovorans (3%), Acinetobacter sp. (5%) Aminobacter sp., (3%) Bacillus sp. (3%), Thauera sp., (3%) and Cytophaga sp. (3%). Relative species distributions among reactor 2 profiled isolates were more evenly distributed between Pseudoxanthomonas (32%), Thauera sp (24%), Acinetobacter (24%), Citrobacter sp (8%), Lactococcus lactis (5%), Lysinibacillus (5%) and Elizabethkingia (2%). In both reactors Gammaproteobacteria dominated the cultured isolates. Culture independent 16S rRNA gene analyses revealed differing profiles for both reactors. Reactor 1 clone distribution was as follows; Zooglea resiniphila (83%), Zooglea oryzae (2%), Pedobacter composti (5%), Neissericeae sp. (2%) Rhodobacter sp. (2%), Runella defluvii (3%) and Streptococcus sp. (3%). RFLP based species distribution among the reactor 2 clones was as follows; Runella defluvii (50%), Zoogloea oryzae (20%), Flavobacterium sp. (9%), Simplicispira sp. (6%), Uncultured Sphingobacteria sp. (6%), Arcicella (6%) and Leadbetterella bysophila (3%). Betaproteobacteria dominated the 16S rRNA gene clones identified in both reactors. FISH analysis with Nile Blue dual staining resolved these divergent findings, identifying the Betaproteobacteria as dominant PHA accumulators within the reactor sludges, although species/strain specific allocations could not be made. GC analysis of the sludge had indicated the presence of both medium chain length as well short chain length PHAs accumulating in both reactors. In addition the cultured isolates from the reactors had been identified previously as mcl and scl PHA producers, respectively. Characterisations of the PHA monomer profiles of the individual isolates were therefore performed to screen for potential novel scl-mcl PHAs. Nitrogen limitation driven PHA accumulation in E2 minimal media revealed a greater propensity among isoates for mcl-pHA production. HPLC analysis indicated that PHB production was not a major feature of the reactor isolates and this was supported by the low presence of scl phaC1 genes among PCR screened isolates. A high percentage distribution of phaC2 mcl-PHA synthase genes was recorded, with the majority sharing high percentage homology with class II synthases from Pseudomonas sp. The common presence of a phaC2 homologue was not reflected in the production of a common polymer. Considerable variation was noted in both the monomer composition and ratios following GC analysis. While co-polymer production could not be demonstrated, potentially novel synthase substrate specificities were noted which could be exploited further in the future.

Tóxicos veiculados por alimentos - diapositivos de apoio às aulas

Caracterização dos acidentes de intoxicação alimentar provocados por constituintes naturais dos alimentos e por ingestão de alimentos contaminados: - Toxinas de origem natural: produzidas por vegetais; produzidas por cogumelos; micotoxinas; substâncias tóxicas produzidas por animais; ficotoxinas; aminas biogénicas; toxinas de origem bacteriana;bactérias, vírus e parasitas patogénicos veiculados por alimentos. - Metais pesados. - Fitofármacos e medicamentos de uso veterinário; - Substâncias que resultam de processos de fabrico, conservação ou preparação dos alimentos.

Laccase bio-grafting a versatile modification tool from green chemistry technologies

Today more than 99% of plastics are petroleum-based because of availability and cost of the raw material. The durability of these disposed plastics contributes to the environmental problems as waste and their persistence in the environment causes deleterious effects on the ecosystem. Environmental pollution awareness and the demand for green technology have drawn considerable attention of both academia and industry into biodegradable polymers. In this regard green chemistry technology has the potential to provide solution to this problematic issue. Laccase bio-grafting has recently been the focus of green chemistry technologies due to the growing environmental concerns, legal restrictions and increasing availability of scientific knowledge. In the last several years, research covering various applications of laccases has been increased rapidly particularly in the field of grafting. In principle, laccase-assisted graft co-polymerization may impart a variety of new functionalities to a polymer. The modified polymers through grafting have a bright future and their development is practically boundless. In present work, novel biodegradable graft copolymers combining the advantages of bacterial cellulose backbone and PHB side chains will be prepared by introducing enzymatic grafting technique. The present research will be a first step in the biopolymer modification. To date no report has been found in literature explaining the enzymatic grafting of PHAs. The technique would also provide an efficient modulation approach to improve the biodegradability and biocompatibility of the graft copolymer. The newly grafted copolymers will exhibit unique functionalities with wider range of potential applications mainly in tissue engineering, biosensors, pharmaceutical industry (drug delivery systems) and bio-plastics.

Optimization of bioplastics production from cheese whey

Dissertation presented in partial fulfillment of the requirements for the degree of Master in Biotechnology