Cristiana A.V. Torres

Polyhydroxyalkanoate (PHA) production by a mixed microbial culture using sugar molasses: Effect of the influent substrate concentration on culture selection

In Polyhydroxyalkanoate (PHA) production processes using Mixed Microbial Culture (MMC), the success of the culture selection step determines, to a great extent, the PHA accumulation performance obtained in the final PHA production stage. In this study, the effect of the influent substrate concentration (30-60Cmmol VFA/L) on the selection of a PHA-storing culture using a complex feedstock, fermented sugar molasses, was assessed. At 30 and 45Cmmol VFA/L, substrate concentration impacted on the process kinetics through a substrate dependent kinetic limitation effect. However, further increasing the carbon substrate concentration to 60Cmmol VFA/L, resulted in an unforeseen growth limitation effect associated with a micronutrient deficiency of the fermented feedstock (magnesium) and high operating pH. Struvite precipitation caused a nutrient limitation which prevented biomass concentration increase, thus causing the feast to famine length ratio to vary in the selection reactor, with subsequent impact on the selective pressure for PHA-storing organisms. A highly dynamic response of the selected population to transient conditions of feast to famine ratio, in the range of 0.21-1.1, was observed. Kinetic (limiting concentration of carbon source) and physiological (loss of internal growth limitation due to the shorter length of famine phase) effects, resulting from variation of the influent substrate concentration, were subsequently demonstrated in batch studies. The culture selected at an influent substrate concentration of 45Cmmol VFA/L showed the best PHA-storing capacity since neither substrate concentration nor feast to famine ratio were limiting factors. This culture, highly enriched in PHA-storing organisms (88%), reached a maximum PHA content of 74.6%.

Kinetics of production and characterization of the fucose-containing exopolysaccharide from Enterobacter A47

A fucose-containing exopolysaccharide (EPS) was produced by the bacterium Enterobacter A47 using glycerol byproduct from the biodiesel industry. The analysis of kinetic data suggested a partially growth associated EPS synthesis model. Although the EPS was composed of fucose, galactose and glucose at all cultivation stages, their relative proportion has varied considerably during the run. At the beginning (24h), glucose was the main component (82.4 wt.%), being fucose and galactose minor components (5.0 wt.% and 10.9 wt.%, respectively), while at the end (96 h) it was composed of 26.0 wt.% fucose, 28.9 wt.% galactose and 43.7 wt.% glucose. The acyl groups content and composition have also changed, reaching their maximum content (19.2wt.%) at the end of the run. Moreover, the molecular weight has increased linearly during the run (from 8×10(5) to 5×10(6)). The changes observed in EPS composition and molecular weight have also had an impact upon the polymers intrinsic viscosity, as shown by its linear increase from 3.95 to 10.72 dL g(-1). The results suggest that the culture might have synthesized at least two distinct EPS, with different sugar composition and average molecular weight, which predominated at different cultivation stages.

Development and characterization of bilayer films of FucoPol and chitosan

Bilayer films of FucoPol and chitosan were prepared and characterized in terms of optical, morphologic, hygroscopic, mechanical and barrier properties, to evaluate their potential application in food packaging. Bilayer films have shown dense and homogeneous layers, and presented enhanced properties when comparing to monolayer FucoPol films. Though, a high swelling degree in contact with liquid water (263.3%) and a high water vapour permeability (0.75×10(-11)mol/msPa), typical of polysaccharide films, was still observed. However, they presented a low permeability to O2 and CO2 (0.47×10(-16)molm/m(2)sPa and 5.8×10(-16)molm/m(2)sPa, respectively). Tensile tests revealed a flexible and resistant film with an elongation at break of 38% and an elastic modulus of 137MPa. The studied properties, in particular the excellent barrier to gases, impart these bilayer films potential to be used in packaging of low moisture content products, as well as in multilayered hydrophobic/hydrophilic/hydrophobic barriers for food products with a broader range of water content.

Study of the interactive effect of temperature and pH on exopolysaccharide production by Enterobacter A47 using multivariate statistical analysis

Enterobacter A47 synthesizes fucose-containing exopolysaccharides (EPS). Maximum EPS production (>7.00 g L(-1)) was obtained for temperature and pH within 25-35°C and 6.0-8.0, respectively. Under these conditions, the polymers contained over 30% fucose. Glucose, galactose, and glucuronic acid contents were about 28%, 25%, and 10%, respectively, and the total acyl groups content was about 20 wt.%. The average molecular weight (Mw) was around 4.0 × 10(6). Outside the optimal temperature and pH ranges, fucose, galactose and glucuronic acid, and the total acyl group contents were reduced, while the glucose content increased, new monomers (rhamnose and glucosamine) were detected, and the Mw increased to ≥ 1.10 × 10(7). This study revealed the ability of Enterobacter A47 to synthesize different heteropolysaccharides as a function of pH and temperature, a feature that can be exploited to obtain tailored polymer composition. Moreover, the production of high fucose content EPS was stable for wide pH and temperature ranges, which is important for the envisaged industrial development of the bioprocess.

Biodegradable films produced from the bacterial polysaccharide FucoPol

FucoPol, an exopolysaccharide produced by Enterobacter A47, grown in bioreactor with glycerol as carbon source, was used with citric acid to obtain biodegradable films by casting. The films were characterized in terms of optical, hygroscopic, mechanical and barrier properties. These films have shown to be transparent, but with a brown tone, imparting small colour changes when applied over coloured surfaces. They were hydrophilic, with high permeability to water vapour (1.01×10(-11)mol/msPa), but presented good barrier properties to oxygen and carbon dioxide (0.7×10(-16)molm/m(2)sPa and 42.7×10(-16)molm/m(2)sPa, respectively). Furthermore, films have shown mechanical properties under tensile tests characteristic of ductile films with high elongation at break, low tension at break and low elastic modulus. Although the obtained results are promising, films properties can be improved, namely by testing alternative plasticizers, crosslinking agents and blends with other biopolymers. Taking into account the observed ductile mechanical properties, good barrier properties to gases when low water content is used and their hydrophilic character, it is foreseen a good potential for FucoPol films to be incorporated as inner layer of a multilayer packaging material.

Influence of temperature on the rheological behavior of a new fucose-containing bacterial exopolysaccharide

The effect of temperature on the rheology of a new fucose-containing extracellular polysaccharide (EPS) was evaluated. The steady state data revealed a shear-thinning behavior, with the viscosity being immediately recovered when the shear rate was decreased. The mechanical spectra indicated viscous solutions with entangled polymer molecules in the range of temperatures studied (from 15 °C to 65 °C). In addition, the Time-Temperature Superposition principle was successfully applied and the Cox-Merz rule was valid, reinforcing the idea of a thermorheologically simple behavior for the EPS in aqueous solution. Furthermore, the viscous and viscoelastic properties at 25 °C were maintained after consecutive heating and cooling cycles, indicating a good thermal stability under temperature fluctuations.

Exopolysaccharide production by a marine Pseudoalteromonas sp. strain isolated from Madeira Archipelago ocean sediments.

Exopolysaccharides (EPS) are polymers excreted by some microorganisms with interesting properties and used in many industrial applications. A new Pseudoalteromonas sp. strain, MD12-642, was isolated from marine sediments and cultivated in bioreactor in saline culture medium containing glucose as carbon source. Its ability to produce EPS under saline conditions was demonstrated reaching an EPS production of 4.4g/L within 17hours of cultivation, corresponding to a volumetric productivity of 0.25g/Lh, the highest value so far obtained for Pseudoalteromonas sp. strains. The compositional analysis of the EPS revealed the presence of galacturonic acid (41-42mol%), glucuronic acid (25-26mol%), rhamnose (16-22mol%) and glucosamine (12-16mol%) sugar residues. The polymer presents a high molecular weight (above 1000kDa). These results encourage the biotechnological exploitation of strain MD12-642 for the production of valuable EPS with unique composition, using saline by-products/wastes as feedstocks.

Rheological studies of the fucose-rich exopolysaccharide FucoPol

In this work, the solution properties of the bacterial fucose-rich polysaccharide, FucoPol, were studied. The effect of pH (3.5-10.0) and ionic strength (0.02-1.0 M NaCl) on the intrinsic viscosity and steady shear flow were evaluated using a central composite rotatable design of experiments and surface response methodology. FucoPols intrinsic and apparent viscosities presented a quite low variation under a wide range of pH (3.5-8.0) and ionic strength (0.05-0.50 M NaCl) values. FucoPol produced viscous solutions with shear-thinning behavior at different polymer concentrations (0.2-1.2 wt.%). Flow curves were successfully described by the Cross model. The viscosity of the first Newtonian plateau varied from 0.01 to 2.47 Pas for polymer concentrations from 0.2 to 1.2 wt.%, and the dependence of the estimated relaxation time with polymer concentration suggests a large degree of interaction between FucoPol molecules. Given the results obtained, FucoPol is proposed as thickening agent for applications in which stability of the apparent viscosity under pH and ionic strength variations is required.

Engineering aspects of microbial exopolysaccharide production

Although the ability to secrete exopolysaccharides (EPS) is widespread among microorganisms, only a few bacterial (e.g. xanthan, levan, dextran) and fungal (e.g. pullulan) EPS have reached full commercialization. During the last years, other microbial EPS producers have been the subject of extensive research, including endophytes, extremophiles, microalgae and Cyanobacteria, as well as mixed microbial consortia. Those studies have demonstrated the great potential of such microbial systems to generate biopolymers with novel chemical structures and distinctive functional properties. In this work, an overview of the bioprocesses developed for EPS production by the wide diversity of reported microbial producers is presented, including their development and scale-up. Bottlenecks that currently hinder microbial EPS development are identified, along with future prospects for further advancement.

Bacterial polymers as materials for the development of micro/nanoparticles

ABSTRACT Bacterial polysaccharides and polyhydroxyalkanoates present physical and chemical characteristics that impart them diverse functional properties, including the ability to produce structures from nano- to macroscale (e.g., spheres, capsules, beads). Such structures may be specially designed to fulfill the requirements of specific applications in different areas, either alone or conjugated with other polymers by means of ionic interactions, hydrogen bonding, or chemical reactions. The interest on using such biomaterials has been increasing due to their unique functional properties, nontoxicity, biodegradability, and biocompatibility. The fields of application of bacterial polymers-based structures include drug delivery, biomedicine, food products, environment, and agriculture, among others. GRAPHICAL ABSTRACT