Carlos Riquelme

Potential probiotic strains in the culture of the Chilean scallop Argopecten purpuratus (Lamarck, 1819)

Abstract This study reports on the search for naturally occurring bacteria which are able to promote the growth and survival of Argopecten purpuratus larvae by inhibiting the activity of other deleterious bacteria that flourish in hatchery cultures. A total of 506 bacterial isolates, obtained from laboratory and hatchery sources, were evaluated for the production of substances inhibitory to a Vibrio anguillarum -related (VAR) larval pathogen, of which 11 (2.2%) were found positive. One of these strains ( Vibrio sp.), when used as a pre-treatment, protected the scallop larvae against subsequent experimental infection with the VAR pathogen.

Selective incorporation of bacteria by Argopecten purpuratus larvae: implications for the use of probiotics in culturing systems of the Chilean scallop.

Abstract Experiments on ingestion rates, colonization and impact of inhibitory producer substances bacteria (IPB) on larvae of Argopecten purpuratus , were carried out to evaluate potential use of bacteria as probiotics in cultures of this Chilean scallop. Three selected strains, named as 11, C33 and 77, obtained from larval cultures of A. purpuratus were tested at different concentrations and incubation times. After 6 h of incubation at a concentration of 10 6 cells ml −1 , A. purpuratus larvae ingested cells of strains 11 and 77, but not those of C33. When comparing bacterial incorporation among these strains, the 77 became the dominant bacteria of the larval microflora, causing no differences in larval survival at different bacterial concentrations. Our results suggest that strain 77 appears as a potential probiotic for scallop larvae and hence, as a promising method to control and prevent infections in hatcheries systems.

Addition of inhibitor-producing bacteria to mass cultures of Argopecten purpuratus larvae (Lamarck, 1819)

Abstract Assays were carried out to test the feasibility of adding inhibitor-producing bacteria (IPB) to mass cultures of Argopecten purpuratus larvae with the aim of replacing the use of antibiotics to minimize deleterious effects of pathogenic bacteria in these cultures. Previously identified IPB (Vibrio sp. C33 and Pseudomonas sp. 11) and a newly identified Bacillus sp. (strain B2) were employed in both summer and winter seasons in a commercial hatchery in northern Chile (23°40′S). Results demonstrated that addition of the selected bacterial cultures did not adversely affect the scallop larvae. In all tests, addition of IPB permitted completion of the pelagic larval phase without the use antibiotic treatment. Observations were also made on changes in the bacterial flora of the larvae upon addition of the IPB strains.

Disinfection of seawater for hatchery aquaculture systems using electrolytic water treatment

A recently marketed electrolytic water treatment system (Hoshizaki) was evaluated for disinfection of seawater used in disease-prone high-intensity aquaculture systems. Bacterial plate counts (CFU), direct bacterial total counts using 4V,6V diamidino-2-phenylindole (DAPI) staining, and viable bacterial total counts using 6-carboxy fluorescein diacetate (6CFDA) showed complete inactivation of bacterial populations at an intensity of z1.3 amp (z2.13 mg Cl l 1 ). This included disinfection of seawater experimentally dosed with the known scallop pathogen Vibrio anguillarum. Experimental use of electrolysis between 1.0 and 4.0 A was able to disinfect cultures of the (food) microalga Isochrysis galbana without deleterious effects on its growth rate. When this technique was applied on a commercial scale in a scallop hatchery, higher microalgal growth rates were achieved compared to those of traditionally autoclaved seawater, or seawater treated with germicidal ultraviolet light (UV). Results suggested that disinfection of hatchery culture waters could be achieved using electrolytic release of very low levels of active Cl ion, providing an effective and economically attractive alternative to currently used methods in these culture systems. D 2002 Elsevier Science B.V. All rights reserved.

Interactions of Botryococcus braunii Cultures with Bacterial Biofilms

Unicellular microalgae generally grow in the presence of bacteria, particularly when they are farmed massively. This study analyzes the bacteria associated with mass culture of Botryococcus braunii: both the planktonic bacteria in the water column and those forming biofilms adhered to the surface of the microalgal cells (∼107–108 culturable cells per gram microalgae). Furthermore, we identified the culturable bacteria forming a biofilm in the microalgal cells by 16S rDNA sequencing. At least eight different culturable species of bacteria were detected in the biofilm and were evaluated for the presence of quorum-sensing signals in these bacteria. Few studies have considered the implications of this phenomenon as regards the interaction between bacteria and microalgae. Production of C4-AHL and C6-AHL were detected in two species, Pseudomonas sp. and Rhizobium sp., which are present in the bacterial biofilm associated with B. braunii. This type of signal was not detected in the planktonic bacteria isolated from the water. We also noted that the bacterium, Rhizobium sp., acted as a probiotic bacterium and significantly encouraged the growth of B. braunii. A direct application of these beneficial bacteria associated with B. braunii could be, to use them like inoculants for large-scale microalgal cultures. They could optimize biomass production by enhancing growth, particularly in this microalga that has a low growth rate.

Transcriptomic response of Argopecten purpuratus post-larvae to copper exposure under experimental conditions.

Few studies have described the molecular response of mollusk larvae to heavy metal exposure. We investigated the response of Chilean scallop Argopecten purpuratus post-larvae to copper exposure under experimental conditions. Post-larvae were maintained with and without copper stress in tanks containing netlon collectors with biofilms that were formed by the bacterium Halomonas sp. and the diatom Amphora sp., known to increase larval settlement. We focused on the analysis of the differential expression patterns of genes associated with copper response. A suppression subtractive hybridization method was used to identify copper-specific up- and down-regulated genes in the post-larvae following 4 days and 8 days exposure to 2.5 and 10 microg/l Cu(+2). This method revealed 145 different sequences corresponding to 10 major physiological functions. The expression of 15 potentially regulated genes was analyzed by real-time PCR in post-larvae at different sampling times during the copper stress. The genes chosen were alpha tubulin, elongation factor 1-alpha (EF1A), tributylin binding protein type 1 (TBT), NADH dehydrogenase subunit 2 (NADH2), cavortin, glutathione peroxidase (GPx), ferritin, heat shock protein 70 (HSP70), Pam highwire rpm1 (Phr1), insulin-like growth factor 1 (IGF1), cellulase, and ribosomal proteins: L18, L44, S3a and S15. This study contributes to the characterization of potential genetic markers that could be used in future environmental monitoring and to explore new mechanisms of stress tolerance in marine mollusk species, especially in early stages of development.

Use of Cellulolytic Marine Bacteria for Enzymatic Pretreatment in Microalgal Biogas Production

ABSTRACT In this study, we designed and evaluated a microalgal pretreatment method using cellulolytic bacteria that naturally degrades microalgae in their native habitat. Bacterial strains were isolated from each of two mollusk species in a medium containing 1% carboxymethyl cellulose agar. We selected nine bacterial strains that had endoglucanase activity: five strains from Mytilus chilensis, a Chilean mussel, and four strains from Mesodesma donacium, a clam found in the Southern Pacific. These strains were identified phylogenetically as belonging to the genera Aeromonas, Pseudomonas, Chryseobacterium, and Raoultella. The cellulase-producing capacities of these strains were characterized, and the degradation of cell walls in Botryococcus braunii and Nannochloropsis gaditana was tested with “whole-cell” cellulolytic experiments. Aeromonas bivalvium MA2, Raoultella ornithinolytica MA5, and Aeromonas salmonicida MC25 degraded B. braunii, and R. ornithinolytica MC3 and MA5 degraded N. gaditana. In addition, N. gaditana was pretreated with R. ornithinolytica strains MC3 and MA5 and was then subjected to an anaerobic digestion process, which increased the yield of methane by 140.32% and 158.68%, respectively, over that from nonpretreated microalgae. Therefore, a “whole-cell” cellulolytic pretreatment can increase the performance and efficiency of biogas production.

Interacción bacteria-microalga en el ambiente marino y uso potencial en acuicultura

El presente estudio tiene como objetivo revisar el conocimiento generado sobre el rol que juegan las interacciones bacteria-microalga en ambientes marinos y dulceacuicolas, definiendo las posibles aplicaciones que puede tener el conocimiento de estas interacciones en el manejo de las aguas costeras y sistemas acuicolas. Los antecedentes proporcionados en este analisis permiten sugerir que bacterias y/o microalgas, constituyen una alternativa para el control de proliferaciones de bacterias y fitoplancton causantes de efectos daninos en ambientes naturales y sistemas cerrados de cultivo. Ademas, las interacciones especificas entre bacteria-microalga permitiria la optimizacion de sistemas productivos en la industria acuicola. Sin embargo, los mecanismos de estas interacciones son pobremente entendidos. Futuras investigaciones debieran ser dirigidas a comprender el modo de accion de las interacciones bacteria-microalga a nivel molecular

Bacteriology of the scallop Argopecten purpuratus (Lamarck, 1819) cultured in Chile

Abstract In this investigation bacteriological analyses of the early developmental stages of Argopecten purpuratus were performed on scallops spawned in situ (hatchery), and in the laboratory under sterile conditions. The results confirmed the occurrence of vertical bacterial transmission. In spawning in the laboratory Pseudomonas, Moraxella and Vibrio were found as the predominant Genera. Subsequently, the effect of these strains on larval survival was tested. From a total of 21 bacterial strains tested the major inhibition of swimming larvae were produced by Vibrio (Strain M); Pseudomonas ; (Strain I) and Moraxella (strains T y S), which caused 96.6%; 81.4%; 61.7% and 51.28% of settlement of the larvae to the bottom, respectively. In a broodstock conditioning experiment, the use of filtered seawater decreased the bacterial concentration in reproductive organs. The lowest total culturable bacteria and vibrios concentration was detected in organisms treated with filtered seawater (0.5 μm).

Inhibition of attachment of some fouling diatoms and settlement of Ulva lactuca zoospores by film-forming bacterium and their extracellular products isolated from biofouled substrata in Northern Chile

The biofouling of surfaces submerged in the marine environment includes primary colonization of the substrate by microorganisms including bacteria, microalgae, and microscopic reproductive propagules of macroorganisms such as algal zoospores. The present study reports the evaluation of the inhibitory potential of biofilms and extracellular products (EP) of the indigenous bacterium Alteromonas sp strain Ni1-LEM on the settlement of marine biofouling such as: (i) eight marine benthic diatoms and (ii) zoospores of the alga Ulva lactuca , as well as the germination of these zoospores and was compared with reference strains with proven antifouling properties, Halomonas marina (ATCC 25374) and Pseudoalteromonas tunicata . Highest antifouling activity was found for the indigenous strain. In attempts to better define the chemical nature of the antifouling substance in the EP of the Alteromonas sp strain Ni1-LEM, the culture filtrates were tested for activity after heat treatment, enzymatic treatments, dialysis through semipermeable membranes, and separation into polar (aqueous) and non-polar (organic) fractions. The results suggested that the antifouling substance in the culture filtrates to be protein or peptide in nature, thermostable, hydrophilic, and equal to or greater than 3500 daltons in molecular size. Antifouling substances from bacteria may lead to the development of novel antifouling agents in the future.