Alberto Bertucco

Microbial inactivation by high-pressure

Abstract High-pressure treatments are receiving a great deal of attention for the inactivation of micro-organisms in foodstuff processing, pressure instead of temperature is used as stabilizing factor. In this context, high hydrostatic pressure treatment is the most studied alternative process, many works reported successful results in inactivating a wide range of micro-organisms under different operative conditions such as temperature, cycles of pressure, exposure time. Furthermore, a number of processes using high pressure treatment (HPT) has already been put into the market. Nevertheless this new technology presents the main limitation to be very expensive and difficult to control and manage because of the extremely high pressure employed, so that the widespread industrial diffusion in industry field appears cumbersome. The treatment with supercritical CO 2 could become a relevant alternative to HPT in the field of microbial inactivation of food as well as an innovative technique for the sterilization of thermally and hydrolytically sensitive polymeric materials in biomedical applications, such as polymeric particles for drug delivery or polymeric implants. It has been demonstrated that the effect of microbial inactivation assuring healthy food preservation is already consistent at pressures moderated (lower than 200 bar) when compared with those employed by traditional hydrostatic-pressure HPT methods (2000–7000 bar). In this work the anti-microbial potential of compressed CO 2 was investigated against gram-negative bacteria, gram-positive bacteria and spores; as model species, Pseudomonas aeruginosa , Bacillus subtilis and spores of B. subtilis were used. The experiments were performed in a semi-continous apparatus at different but mild operative conditions. Excellent results were obtained for micro-organisms, under appropriate conditions the survival ratio of bacteria could be reduced to about seven orders of magnitude. Inactivation of spores under the same conditions, found to be conflicting in open literature, was not satisfactory. Spore inactivation was possible by coupling combination of higher temperature and longer contact time conditions. The application of pressure cycles was also found to be beneficial.

Adjusted Light and Dark Cycles Can Optimize Photosynthetic Efficiency in Algae Growing in Photobioreactors

Biofuels from algae are highly interesting as renewable energy sources to replace, at least partially, fossil fuels, but great research efforts are still needed to optimize growth parameters to develop competitive large-scale cultivation systems. One factor with a seminal influence on productivity is light availability. Light energy fully supports algal growth, but it leads to oxidative stress if illumination is in excess. In this work, the influence of light intensity on the growth and lipid productivity of Nannochloropsis salina was investigated in a flat-bed photobioreactor designed to minimize cells self-shading. The influence of various light intensities was studied with both continuous illumination and alternation of light and dark cycles at various frequencies, which mimic illumination variations in a photobioreactor due to mixing. Results show that Nannochloropsis can efficiently exploit even very intense light, provided that dark cycles occur to allow for re-oxidation of the electron transporters of the photosynthetic apparatus. If alternation of light and dark is not optimal, algae undergo radiation damage and photosynthetic productivity is greatly reduced. Our results demonstrate that, in a photobioreactor for the cultivation of algae, optimizing mixing is essential in order to ensure that the algae exploit light energy efficiently.

Acclimation of Nannochloropsis gaditana to different illumination regimes: Effects on lipids accumulation

Algae are interesting potential sources of biodiesel, although research is still needed to develop efficient large scale productions. One major factor affecting productivity is light use efficiency. The effect of different light regimes on the seawater alga Nannochloropsis gaditana was accessed monitoring growth rate and photosynthetic performances. N. gaditana showed the capacity of acclimating to different light intensities, optimizing its photosynthetic apparatus to illumination. Thanks to this response, N. gaditana maintained similar growth rates under a wide range of irradiances, suggesting that this organism is a valuable candidate for outdoor productions in variable conditions. In the conditions tested here, without external CO(2) supply, light intensity alone was not found to be a major signal affecting lipids accumulation showing the absence of a direct regulatory link between the light stress and lipids accumulation. Strong illumination can nevertheless indirectly influences lipid accumulation if combined with other stresses or in the presence of excess CO(2).

Production of micronic particles of biocompatible polymer using supercritical carbon dioxide

Three micronization techniques, based on the use of supercritical carbon dioxide, were investigated to produce microspheres of a natural biocompatible polysaccharide. Particles smaller than 20 mum were obtained by means of the rapid expansion of a supercritical solution method (RESS), both with and without cosolvents. The mean diameter of the particles was reduced to about 0.5 mum when a solution of the polymer in an organic solvent was expanded by using carbon dioxide as a supercritical antisolvent (SAS). The SAS process was operated both in a continuous and in a batch mode. The former leads to aggregated structures and fibers, and the latter to the formation of micronic spherical particles. It was found that the experimental temperature did not substantially affect the shape and dimension of the particles. A stronger dependence is shown with respect to the solute concentration in the starting solution. The proposed method is attractive as the basis of a new process for the preparation of drug delivery systems. (c) 1997 John Wiley & Sons, Inc.

Excess CO2 supply inhibits mixotrophic growth of Chlorella protothecoides and Nannochloropsis salina

Mixotrophy can be exploited to support algal growth over night or in dark-zones of a photobioreactor. In order to achieve the maximal productivity, however, it is fundamental also to provide CO(2) in excess to maximize photosynthetic activity and phototropic biomass production. The aim of this paper is to verify the possibility of exploiting mixotrophy in combination with excess CO(2). Two species with high biomass productivity were selected, Nannochloropsis salina and Chlorella protothecoides. Different organic substrates available at industrial scale were tested, and glycerol chosen for its ability to support growth of both species. In mixotrophic conditions, excess CO(2) stimulated photosynthesis but blocked the metabolization of the organic substrate, thus canceling the advantages of mixotrophy. By cultivating microalgae under day-night cycle, organic substrate supported growth during the night, but only if CO(2) supply was not provided. This represents thus a possible method to reconcile CO(2) stimulation of photosynthesis with mixotrophy.

Inactivation of Bacillus subtilis spores by supercritical CO2 treatment

Bacillus subtilis spores were suspended in saline solution (107 cfu/ml) and treated by both conventional heating and CO2 batch treatment at an operating pressure in the range of 70–150 bar under identical temperature conditions. Temperatures tested were in the range of 36–75 °C. Survival curves indicated significantly higher lethality when spores were treated with supercritical CO2 (SC-CO2) rather than with heating alone. These results appear particularly evident at 60 °C, a temperature at which conventional heating gave no spore-inactivation after a treating time as long as 24 h, whereas a 6 h SC-CO2 treatment led to complete sterilization. At 75 °C spores were partially killed with conventional heating but a treatment of 2 with SC-CO2 hours assured total inactivation. It is concluded that spore-inactivation during SC-CO2 treatment was only in part due to thermal effect (at the higher temperature of 75 °C) and there was a significant additional effect caused by CO2 penetration inside the latent bacteria forms.

Production of Solid Lipid Submicron Particles for Protein Delivery Using a Novel Supercritical Gas‐Assisted Melting Atomization Process

A supercritical carbon dioxide micronization technique based on gas-assisted melting atomization has been designed to prepare protein-loaded solid lipid submicron particles. The supercritical process was applied to homogeneous dispersions of insulin in lipid mixtures: (1) tristearin, Tween-80, phosphatidylcholine and 5 kDa PEG (1:0.1:0.9:1 and 1:0.1:0.9:2 weight ratio); and (2) tristearin, dioctyl sulfosuccinate and phosphatidylcholine (1:1:0.5 weight ratio). Optimized process conditions yielded dry nonagglomerated powders with high product recovery (70%, w/w). Dynamic light scattering and transmission electron microscopy showed that two size fractions of particles, with 80-120 and 200-400 nm diameters, were produced. In all final products, dimethylsulfoxide used to prepare the insulin/lipid mixture was below 20 ppm. Protein encapsulation efficiency increased up to 80% as the DMSO content in the insulin/lipid mixture increased. Compared to the particles without PEG, the polymer-containing particles dispersed rapidly in water, and the dispersions were more stable under centrifugation as less than 20% of suspended particles precipitated after extensive centrifugation. In vitro, the protein was slowly released from the formulation without PEG, while a burst and faster release were obtained from the formulations containing PEG. Subcutaneous injection to diabetic mice of insulin extracted from the particles showed that the supercritical process did not impair the protein hypoglycemic activity.

Development of a composition estimator for binary distillation columns. Application to a pilot plant

Abstract A nonlinear extended Kalman filter, which infers the compositions of the streams leaving a binary distillation column from temperature measurements, is developed. The accuracy of the distillation column model on which the estimator is based, is discussed in connection with the reliability of the obtained estimates. The estimator performance is checked by comparison with the dynamic behavior of a distillation pilot plant, where the separation of a binary mixture of ethanol and water takes place.

A recirculation apparatus for vapor-liquid equilibrium measurements of refrigerants. Binary mixtures of R600a, R134a and R236fa

Abstract An apparatus for the measurement of vapor–liquid equilibria (P–T–x–y) data of refrigerant systems was designed, built and tested. The recirculation method was used and the vapor phase was forced through the liquid phase by a magnetic pump. The thermodynamic equilibrium was reached in a visual cell with an internal volume of 50 cm3. The compositions of the phases were analyzed by a gas chromatograph connected on-line with either FID or TCD detectors. The operating temperature and pressure in the apparatus, as tested before delivery to the laboratory [R. Stryjek, personal communication], range from 240 to 350 K and from 0.1 to 10 MPa, respectively. The estimated accuracy of the measured data was ±0.01 K for temperature, ±0.8 kPa for pressure and 0.001 in mole fraction for liquid and vapor compositions. Vapor–liquid equilibria data were obtained for the following systems: 1,1,1,2-tetrafluorethane+isobutane (R134a+R600a) at 293.66 and 303.68 K; 1,1,1,2-tetrafluorethane+1,1,1,3,3,3-hexafluoropropane (R134a+R236fa) at 283.62 and 303.68 K; and isobutane+1,1,1,3,3,3-hexafluoropropane (R600a+R236fa) at 303.68 K. The thermodynamic consistency of the experimental results was tested.

Biofuels from microalgae: lipid extraction and methane production from the residual biomass in a biorefinery approach.

Renewable fuels and energy are of major concern worldwide and new raw materials and processes for its generation are being investigated. Among these raw materials, algae are a promising source of lipids and energy. Thus, in this work four different algae have been used for lipid extraction and biogas generation. Lipids were obtained by supercritical CO2 extraction (SCCO2), while anaerobic digestion of the lipid-exhausted algae biomass was used for biogas production. The extracted oil composition was analyzed (saturated, monounsaturated and polyunsaturated fatty acids) and quantified. The highest lipid yields were obtained from Tetraselmis sp. (11%) and Scenedesmus almeriensis (10%), while the highest methane production from the lipid-exhausted algae biomass corresponded to Tetraselmis sp. (236mLCH4/gVSadded).