Alternatives to classic solvents for the isolation of bioactive compounds from Chrysochromulina rotalis.

This paper demonstrates a sequential partitioning method for isolating bioactive compounds from Chrysochromulina rotalis using a polarity gradient, replacing classic and hazardous solvents with greener alternatives. Seventeen solvents were evaluated based on their Hansen solubility parameters and for having a similar polarity to the solvents they would replace, four of which were selected as substitutes in the classic fractionation process. Considering the fatty acid and carotenoid recovery yields obtained for each of the solvents, it has been proposed to replace hexane (HEX), toluene (TOL), dichloromethane (DCM) and n-butanol (BUT) with cyclohexane, chlorobenzene, isobutyl acetate and isoamyl alcohol, respectively. In addition, cytotoxic activity was observed when the TOL and DCM solvent extracts were tested against tumour cell lines, demonstrating the antiproliferative potential of compounds containing, for example, fucoxanthin, fatty acids, peptides, isoflavonoids or terpenes, among others.

An optimisation approach for culturing shear-sensitive dinoflagellate microalgae in bench-scale bubble column photobioreactors.

The dinoflagellate Karlodinium veneficum was grown in bubble column photobioreactors and a genetic algorithm-based stochastic search strategy used to find optimal values for the culture parameters gas flow rate, culture height, and nozzle sparger diameter. Cell production, concentration of reactive oxygen species (ROS), membrane fluidity and photosynthetic efficiency were studied throughout the culture period. Gas-flow rates below 0.26Lmin(-1), culture heights over 1.25m and a nozzle diameter of 1.5mm were found to provide the optimal conditions for cell growth, with an increase of 60% in cell production with respect to the control culture. Non-optimal conditions produced a sufficiently high shear stress to negatively affect cell growth and even produce cell death. Cell physiology was also severely affected in stressed cultures. The production of ROS increased by up to 200%, whereas cell membrane fluidity decreased by 60% relative to control cultures. Photosynthetic efficiency decreased concomitantly with membrane fluidity.

Protein production using the baculovirus-insect cell expression system.

The baculovirus-insect cell expression system is widely used in producing recombinant proteins. This review is focused on the use of this expression system in developing bioprocesses for producing proteins of interest. The issues addressed include: the baculovirus biology and genetic manipulation to improve protein expression and quality; the suppression of proteolysis associated with the viral enzymes; the engineering of the insect cell lines for improved capability in glycosylation and folding of the expressed proteins; the impact of baculovirus on the host cell and its implications for protein production; the effects of the growth medium on metabolism of the host cell; the bioreactors and the associated operational aspects; and downstream processing of the product. All these factors strongly affect the production of recombinant proteins. The current state of knowledge is reviewed.

Mixotrophic growth of Phaeodactylum tricornutum on fructose and glycerol in fed-batch and semi-continuous modes.

Mixotrophic cultures of Phaeodactylum tricornutum were carried out in bubble columns using fructose and glycerol in indoor fed-batch and semi-continuous modes. In the fed-batch cultures, different nutrient-addition strategies, combined with stepwise increments in the light intensity, were assayed. It was found that glycerol promoted significantly higher biomass productivity than fructose. A glycerol-induced photoinhibition that arrested the growth of P. tricornutun was also observed. As this was considered a limitation as regards transferring the fed-batch mode to outdoor conditions, this information was used to culture P. tricornutum in semi-continuous mode. Similar glycerol-induced photoinhibition was not observed in these cultures, even at highest dilution rates. Although the highest biomass (1.5 g L(-1) d(-1)) and EPA (40 mg L(-1) d(-1)) productivities found in the semi-continuous cultures were lower than those obtained photoautotrophically in outdoor photobioreactors, the findings showed that semi-continuous mode was an excellent candidate for transferring mixotrophic culture to an outdoor setting.

Modelling of multi-nutrient interactions in growth of the dinoflagellate microalga Protoceratium reticulatum using artificial neural networks.

This study examines the use of artificial neural networks as predictive tools for the growth of the dinoflagellate microalga Protoceratium reticulatum. Feed-forward back-propagation neural networks (FBN), using Levenberg-Marquardt back-propagation or Bayesian regularization as training functions, offered the best results in terms of representing the nonlinear interactions among all nutrients in a culture medium containing 26 different components. A FBN configuration of 26-14-1 layers was selected. The FBN model was trained using more than 500 culture experiments on a shake flask scale. Garson's algorithm provided a valuable means of evaluating the relative importance of nutrients in terms of microalgal growth. Microelements and vitamins had a significant importance (approximately 70%) in relation to macronutrients (nearly 25%), despite their concentrations in the culture medium being various orders of magnitude smaller. The approach presented here may be useful for modelling multi-nutrient interactions in photobioreactors.

A low-cost culture medium for the production of Nannochloropsis gaditana biomass optimized for aquaculture.

Nannochloropsis gaditana is a microalga with a high nutritional value and a protein and polyunsaturated fatty acid (PUFA) content that makes it interesting as a feed in aquaculture. To maximize its productivity and nutritional value in large-scale culture, a well-known commercial medium was optimized to the most favorable nutrient level using commercial fertilizers. Optimal growth conditions were obtained in the alternative fertilizer-based medium at a nitrogen concentration of 11.3 mM, a phosphorus concentration of 0.16 mM, and a micronutrient concentration of 30 µL L(-1). This alternative medium allowed to obtain a biomass concentration similar to that achieved when using the commercial formula but with a reduction in Cu, Fe, and Mo content of 71%, 89%, and 99%, respectively. A maximum biomass productivity of 0.51 g L(-1) d(-1) was obtained. The eicosapentaenoic acid and protein contents of the biomass were 2.84% and 44% of dry weight, respectively.

Adaptation of the Spodoptera exigua Se301 insect cell line to grow in serum-free suspended culture. Comparison of SeMNPV productivity in serum-free and serum-containing media.

Spodoptera exigua Se301 cells have been successfully adapted to two different commercial serum-free media (SFM; Ex-Cell 420 and Serum-Free Insect Medium-1) by gradually reducing the 10 %-added serum-containing medium content from 100 % to 0 % (v/v) in suspended cultures. Both direct adaptation to a serum-free medium and cell growth in the absence of protective additives against fluid dynamic stress [polyvinyl pyrrolidone and polyvinyl alcohol] and disaggregation [dextran sulfate] proved impossible. Cells grew reproducibly in both SFMs once the serum had been completely removed, although the use of Ex-Cell 420 resulted in higher growth rates and cell densities. Turbulence was sufficiently high to reduce growth rates and final cell densities at the highest Reynolds number investigated, although no clear influence of agitation was observed on virus productivity. Both attached and suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Cells adapted to different conditions (attached or suspended culture, serum-containing or serum-free medium) showed different occlusion bodies productivities at the two multiplicities of infection assayed (0.1 and 0.5).

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640.

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long-term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.

Cultivation of explants of the marine sponge Crambe crambe in closed systems.

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.

Effects of diazepam on fentanyl-induced epileptoid EEG activity and increase of multineuronal firing in limbic and mesencephalic brain structures.

Electroencephalographic and clinical signs of epileptoid activity have been associated with the administration of fentanyl during surgery in patients. These phenomena have been in turn related to changes in metabolic rate, oxygen consumption, and blood flow in specific brain structures both in humans and experimental animals. However, direct evidence showing changes in neuronal firing in specific brain regions during fentanyl-induced epileptoid activity has not been reported. Eight adult male cats with chronically implanted bipolar electrodes in the mesencephalic reticular formation, hippocampus, amygdala, and parieto-occipital cortex were included in the study. Different treatments, i.e., vehicle-fentanyl or diazepam-fentanyl, were administered to the experimental animals at 7-day intervals under neuromuscular blockade and assisted ventilation. Electroencephalographic (EEG) seizures, grouped and isolated spikes, and significant increases of multineuronal activity (MUA) were elicited by fentanyl, 50 micrograms/kg iv, in these brain structures. Both EEG and MUA changes reached their maximal values within 6 min of fentanyl administration, and then diminished as time elapsed. Diazepam, 100, 200, or 400 micrograms/kg, but not 50 micrograms/kg iv, significantly reduced or prevented the fentanyl-induced epileptoid EEG activity and MUA increases. The present results show that both fentanyl-induced epileptoid EEG activity as well as the concomitant increase in MUA of brain subcortical structures are part of the same epileptogenic phenomenon, mainly generated at limbic structures. In addition, the effects of diazepam against both epileptoid EEG activity and increase of MUA of brain subcortical structures support the use of benzodiazepines as premedicants for fentanyl anesthesia in order to prevent or to reduce epileptoid phenomena that can result from opioid administration during the anesthetic procedures.

Effects of propofol on alterations of multineuronal activity of limbic and mesencephalic structures and neurological deficit elicited by acute global cerebral ischemia.

The increment of GABAergic inhibitory activity, the reduction of metabolic rate and oxygen consumption induced by propofol on the neuronal components of brain structures, and its antioxidant potential have supported the possible beneficial effects of this drug against brain damage elicited by cerebral ischemia. Multineuronal activity (MUA) and EEG from mesencephalic reticular formation, hippocampus, and amygdala, and EEG from the parietooccipital cortex were recorded and analyzed during vehicle or propofol, 0.25 mg/kg/min i.v., administered during a 6 h period following a 10 min cardiorespiratory arrest and 2-4 min of reanimation in two groups of cats under neuromuscular blockade and assisted ventilation. This was continued daily during alertness for 8 days after cardiorespiratory arrest along with determining daily neurological deficit scores. Mean MUA frequency, progressively increasing in subcortical structures of untreated cats during the hours following cardiorespiratory arrest, was significantly lower in propofol treated cats. A significant reduction of MUA in the hippocampus was then observed in the untreated but not in the propofol treated cats, and in amygdala in both treated and untreated cats. Alterations of MUA were not observed in the mesencephalic reticular formation during alertness on the days after cardiorespiratory arrest. Significantly lower neurological deficit scores were recorded in propofol treated than in untreated cats the days after cardiorespiratory arrest. It can be concluded that propofol is capable of reducing both brain electrical activity alterations in specific brain structures, and neurological deficit elicited by complete global cerebral ischemia in cats. Inhibition of MUA from limbic and mesencephalic brain structures induced by propofol early after global cerebral ischemia could be related to these effects.