Catarina S. Oliveira

Characterization of polyhydroxyalkanoates synthesized from microbial mixed cultures and of their nanobiocomposites with bacterial cellulose nanowhiskers.

The present work reports on the production and characterization of polyhydroxyalkanoates (PHAs) with different valerate contents, which were synthesized from microbial mixed cultures, and the subsequent development of nanocomposites incorporating bacterial cellulose nanowhiskers (BCNW) via solution casting processing. The characterization of the pure biopolyesters showed that the properties of PHAs may be strongly modified by varying the valerate ratio in the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer, as expected. Increasing the valerate content was seen to greatly decrease the melting temperature and enthalpy of the material, as well as its rigidity and stiffness, resulting in a more ductile behaviour. Additionally, the higher valerate PHA displayed higher permeability to water and oxygen and higher moisture sensitivity. Subsequently, BCNW were incorporated into both PHA grades, achieving a high level of dispersion for a 1 wt.-% loading, whereas some agglomeration took place for 3 wt.-% BCNW. As evidenced by DSC analyses, BCNW presented a nucleating effect on the PHA matrices. BCNW also increased the thermal stability of the polymeric matrices when properly dispersed due to strong matrix-filler interactions. Barrier properties were seen to depend on relative humidity and improved at low nanofiller loadings and low relative humidity.

Response of a three-stage process for PHA production by mixed microbial cultures to feedstock shift: impact on polymer composition.

Polyhydroxyalkanoates (PHA) can be produced by mixed microbial cultures (MMC) using a three-stage process. An attractive feature of MMC for PHA production is the ability to use waste/surplus feedstocks. In this study, the effect of a feedstock shift, mimicking a seasonal feedstock scenario and/or as a strategy for controlling polymer composition, on a MMC PHA production process was assessed using cheese whey (CW) and sugar cane molasses (SCM) as model feedstocks. The acidogenic stage responded immediately to the feedstock shift by changing the fermented products profile, with acetate and butyrate being the main acids produced from CW, while for SCM propionate and valerate were the dominant products. The fermentation process was then quite stable during long term operation. The PHA culture selection stage also responded quickly to the fermented feestocks shift, generating a polymer whose composition was linearly dependent on the concentration of HV and HB precursors produced in the acidogenic stage. The selected culture reached a maximum PHA content of 56% and 65% with fermented SCM and CW, respectively. Mixing fermented CW and SCM, in equal volume proportions, demonstrated the possibility of using different fermented feedstocks for tailoring polymer composition.

Strategies for efficiently selecting PHA producing mixed microbial cultures using complex feedstocks: Feast and famine regime and uncoupled carbon and nitrogen availabilities

Production of polyhydroxyalkanoates (PHAs) by open mixed microbial cultures (MMCs) has been attracting increasing interest as an alternative technology to PHA production by pure cultures, due to the potential for lower costs associated with the use of open systems (eliminating the requirement for sterile conditions) and the utilisation of cheap feedstock (industrial and agricultural wastes). Such technology relies on the efficient selection of an MMC enriched in PHA-accumulating organisms. Fermented cheese whey, a protein-rich complex feedstock, has been used previously to produce PHA using the feast and famine regime for selection of PHA accumulating cultures. While this selection strategy was found efficient when operated at relatively low organic loading rate (OLR, 2g-CODL-1d-1), great instability and low selection efficiency of PHA accumulating organisms were observed when higher OLR (ca. 6g-CODL-1d-1) was applied. High organic loading is desirable as a means to enhance PHA productivity. In the present study, a new selection strategy was tested with the aim of improving selection for high OLR. It was based on uncoupling carbon and nitrogen supply and was implemented and compared with the conventional feast and famine strategy. For this, two selection reactors were fed with fermented cheese whey applying an OLR of ca. 8.5g-CODL-1 (with 3.8g-CODL-1 resulting from organic acids and ethanol), and operated in parallel under similar conditions, except for the timing of nitrogen supplementation. Whereas in the conventional strategy nitrogen and carbon substrates were added simultaneously at the beginning of the cycle, in the uncoupled substrates strategy, nitrogen addition was delayed to the end of the feast phase (i.e. after exogenous carbon was exhausted). The two different strategies selected different PHA-storing microbial communities, dominated by Corynebacterium and a Xantomonadaceae, respectively with the conventional and the new approaches. The new strategy originated a more efficient PHA-production process than the conventional one (global PHA productivity of 6.09g-PHAL-1d-1 and storage yield of 0.96 versus 2.55g-PHAL-1d-1 and 0.86, respectively). Dissociation between the feast to famine length ratio (F/F) and storage efficiency was shown to be possible with the new strategy, allowing selection of an efficient PHA-storing culture with complex feedstock under high organic loading rates.

The Key Role of Sulfation and Branching on Fucoidan Antitumor Activity

There is an urgent need for antitumor bioactive agents with minimal or no side effects over normal adjacent cells. Fucoidan is a marine-origin polymer with known antitumor activity. However, there are still some concerns about its application due to the inconsistent experimental results, specifically its toxicity over normal cells and the mechanism behind its action. Herein, three fucoidan extracts (FEs) have been tested over normal and breast cancer cell lines. From cytotoxicity results, only one of the extracts shows selective antitumor behavior (at 0.2 mg mL-1 ), despite similarities in sulfation degree and carbohydrates composition. Although the three FEs present different molecular weights, depolymerization of selected samples discarded Mw as the key factor in the antitumor activity. Significant differences in sulfates position and branching are observed, presenting FE 2 the higher branching degree. Based on all these experimental data, it is believed that these last two properties are the ones that influence the cytotoxic effects of fucoidan extracts.

Biofunctional nanofibrous substrate comprising immobilized antibodies and selective binding of autologous growth factors

The immobilization of biomolecules at the surface of different biomedical devices has attracted enormous interest in order to enhance their biological functionality at the cellular level. This work aims to develop a biofunctional polymeric substrate capable of selectively binding growth factors (GFs) of interest from a pool of proteins present in a biological fluid: platelet lysate (PL). To achieve this goal, the surface of electrospun PCL nanofibers needs to be activated and functionalized to be able to insert chemical groups for the immobilization of antibodies. After determining the maximum immobilization capacity of each antibody, TGF-β1 (12 μg mL(-1)), bFGF (8 μg mL(-1)), and VEGF (4 μg mL(-1)), the next step was to confirm their bioavailability using recombinant proteins. The binding efficiency of PL-derived GFs was of 84-87% for TGF-β1, 55-64% for bFGF, and 50-59% for VEGF. Cellular assays confirmed the biological activity of the bound VEGF (both recombinant and PL-derived). Multiple antibodies (i.e., bFGF and VEGF) were also immobilized over the same structure in a mixed or side-by-side fashion. Using both autologous biological fluids and cells, it is possible to use this platform to implement very effective and personalized therapies that can be tailored to specific medical conditions.

Kinetic and stoichiometric characterization of a fixed biofilm reactor by pulse respirometry.

An in situ respirometric technique was applied to a sequential biofilm batch reactor treating a synthetic wastewater containing acetate. In this reactor, inoculated with mixed liquor from a wastewater plant, unglazed ceramic tiles were used as support media while maintaining complete mixing regime. A total of 8 kinetic and stoichiometric parameters were determined by in situ pulse respirometry; namely substrate oxidation yield, biomass growth yield, storage yield, storage growth yield, substrate affinity constant, storage affinity constant, storage kinetic constant and maximum oxygen uptake rate. Additionally, biofilm growth was determined from support media sampling showing that the colonization process occurred during the first 40 days, reaching an apparent steady-state afterward. Similarly, most of the stoichiometric and kinetic parameters were changing over time but reached steady values after day 40. During the experiment, the respirometric method allowed to quantify the amount of substrate directed to storage, which was significant, especially at substrate concentration superior to 30mg CODL(-1). The Activated Sludge Model 3 (ASM3), which is a model that takes into account substrate storage mechanisms, fitted well experimental data and allowed confirming that feast and famine cycles in SBR favor storage. These results also show that in situ pulse respirometry can be used for fixed-bed reactors characterization.

Determination of kinetic and stoichiometric parameters of pseudomonas putida F1 by chemostat and In situ pulse respirometry

The applicability of pulse respirometry, for the estimation of kinetic and stoichiometric parameters in pure cultures was evaluated by comparison with traditional chemostat method. Pseudomonas putida F1 was cultured in a continuous stirred tank reactor, using glucose as sole carbon source. The reactor was operated under steady-state with six dilution rates, ranging from 0.06 to 0.35 h-1. Substrate and biomass concentration were measured and used to estimate kinetic and stoichiometric parameters, according to the Monod model. An in situ respirometry method was also applied to the reactor, with the injection of pulses of glucose from 19 to 97 mg L-1. The respirograms obtained were used to estimate the kinetic and stoichiometric parameters according to ASM1 and ASM3 models. No significance difference was observed between parameters estimated by chemostat and respirometric methods. The glucose affinity constant was from 0.4 to 0.7 mg L-1, the maximum specific growth rate was from varying from 0.14 to 0.20 h-1, and the growth yield was from 0.41 to 0.67. These results confirm that in situ pulse respirometry is a suitable method for kinetic and stoichiometric parameters estimation.