José Pintado

Characterization of microbial biomasses and amylolytic preparations obtained from mussel processing waste treatment

This work describes a treatment of glycogen-rich wastes from industrial mussel processing, involving the production of a protein fraction and a medium suitable for the culture of amylolytic microfungi. A strain of Aspergillus oryzae was chosen which allows the simultaneous production of single cell protein and a highly stable amylolytic preparation. The characteristics of the biomass obtained (proportion and in vitro digestibility of the proteins, levels of essential amino acids and nucleic acids, types of fatty acids present) were shown to be suitable, in principle, for animal feeds. The amylolytic preparation, obtained by ultrafiltration (with cut-off at 30 kD) of the cell-free medium, was very similar to commercial α-amylase preparations, containing mainly α-amylase, together with a small proportion of glucoamylase.

Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms

Vibrio species isolated from diseased seahorses were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The results demonstrated that Vibrio alginolyticus and Vibrio splendidus were predominant in the lesions of these seahorses. To our knowledge, this is the first time that these bacterial species have been associated with disease symptoms in captive-bred seahorses.

Optimization of nutrient concentration for citric acid production by solid-state culture of Aspergillus niger on polyurethane foams

Abstract Citric acid production from mussel processing effluents by Aspergillus niger in solid-state culture was studied using polyurethane foam particles soaked with the culture medium. Conditions were used that allowed comparison of the results with those obtained before in submerged culture and the attribution of the differences to the characteristics of solid-state culture. A screening of several strains gave different results than in submerged culture and a reduction in the incubation time. The joint effect of nitrogen and phosphorus initial concentrations was examined by means of orthogonal factorial designs in five selected strains. The results showed that different requirements of N and P for each individual strain were needed. Optimization was done using different experimental strategies for two selected strains. Following either the direction of the linear approximation gradient of the first-order empirical model obtained or with a new rotatable factorial design, an optimum value could be obtained. In comparison with previous results in submerged culture, the strains with high requirements of N and P seemed to be disfavored in solid-state culture. The ones with low requirements were favored and showed a bigger tolerance to a surplus of both nutrients.

Phylogenetic characterization and in situ detection of bacterial communities associated with seahorses (Hippocampus guttulatus) in captivity.

Although there are several studies describing bacteria associated with marine fish, the bacterial composition associated with seahorses has not been extensively investigated since these studies have been restricted to the identification of bacterial pathogens. In this study, the phylogenetic affiliation of seahorse-associated bacteria was assessed by 16S rRNA gene sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rRNA analysis. Both methods revealed that Vibrionaceae was the dominant population in Artemia sp. (live prey) and intestinal content of the seahorses, while Rhodobacteraceae was dominant in water samples from the aquaculture system and cutaneous mucus of the seahorses. To our knowledge, this is the first time that bacterial communities associated with healthy seahorses in captivity have been described.

Lactic acid production from mussel processing wastes with an amylolytic bacterial strain

Abstract The aim of this work was to study the capability of amylolytic lactic acid bacteria to hydrolyse the glycogen in mussel processing wastes (MPW) for lactic acid production. From four strains tested, Lactobacillus plantarum A6 was selected. Cultures of this strain on MRS medium with starch or mussel glycogen, showed similar levels of amylase production for both polysaccharides. With glycogen, sugar consumption rate was lower than with soluble starch and, although the micro-organism produced higher yields of enzyme, a fraction of approximately 20% of the total sugars remained undegraded. However, the different substrates did not seem to induce the production of different types of amylase and the values of the apparent K m for starch and for glycogen were close, revealing a similar affinity for both substrates. Therefore, the differences in hydrolysis and consumption of the substrate seem to be related with the higher grade of branching of glycogen. Culture in MPW showed a decrease in pH unfavourable for amylolytic activity and uncoupling between lactic acid and biomass production. By controlling pH and increasing the initial protein concentration to avoid uncoupling, similar lactic acid production and higher yields than obtained with starch were reached with MPW.

Interactions affecting gibberellic acid production in solid-state culture: A factorial study

Abstract The production of GA3 in solid-state culture was studied using systems composed of maize cob particles soaked in an amylaceous effluent, as a function of three variables-particle diameter, volume of liquid phase, and substrate concentration—with a determinant influence on the response. The empirical model obtained provides an explanation of the behavior of the system in terms of a series of interactions with underlying problems of water activity and mass transfer, that can, however, be manipulated in a simple and reproducible manner. This model also allows the suitable orientation of production improvement, the interpretation of the mechanism of substrate inhibition detected, as well as means of correcting it without reducing the initial concentration of the substrate, and leads to an 11-fold increase in the production obtained in the preliminary assays.

Real-time PCR detection and quantification of fish probiotic Phaeobacter strain 27-4 and fish pathogenic Vibrio in microalgae, rotifer, Artemia and first feeding turbot (Psetta maxima) larvae

Aims:  To develop a SYBR Green quantitative real‐time PCR protocol enabling detection and quantification of a fish probiotic and two turbot pathogenic Vibrio spp. in microcosms.

ON-LINE MONITORING OF CITRIC ACID PRODUCTION IN SOLID-STATE CULTURE BY RESPIROMETRY

The aim of this work was to study the possibility of monitoring citric acid production from mussel processing wastes (MPW) by Aspergillus niger in solid-state culture (SSC) on an inert support. This was conducted by measuring CO2 and O2 concentration in exhaust gases, using an automatic sampler connected to a gas chromatograph and a data acquisition system. The procedure permitted information on the physiological state of the culture to be obtained. A relationship between citric acid accumulation and a decrease in CO2 production was found, allowing citric acid production to be followed in real-time. Moreover, respiratory activity (μr) can be estimated and the effect of different variables on this parameter studied. Initial nitrogen concentration, a critical factor for achieving high production of citric acid from MPW in submerged culture, revealed no effect in SSC. This indicates a tolerance of SSC to higher concentrations of nitrogen constituting an advantage when using residual media with high levels of protein and variability in their composition.

Joint effect of nitrogen and phosphorus concentrations on citric acid production by different strains of Aspergillus niger grown on an effluent

SummaryThe joint effects of the initial concentration of N and P on the production of citric acid by 11 strains of Aspergillus niger on an effluent are studied by means of orthogonal full-factorial 22 designs. The results allow the definition of a series of empirical equations describing well differentiated types of behaviour. This demonstrates the usefulness of the procedure followed in order to establish selection criteria for the more suitable strains, to determine the tendencies that permit approaching optimum production and evaluate the viability of different culture options characterized by discontinuous feeding of one or another nutrient.

Multiple competitive PCR–DGGE as a tool for quantifying and profiling defined mixed cultures of lactic acid bacteria during production of probiotics from complex polysaccharides

Aims: To apply a denaturing gradient gel electrophoretic (DGGE) method to quantify and profile individual strains during a mixed culture fermentation.