Effect of light on ex situ production of symbiotic corals

The increasing interest in coral culture for biotechnological applications, to supply the marine aquarium trade, or for reef restoration programs, has prompted researchers to optimize coral culture protocols, with emphasis to ex situ production. When cultured ex situ, the growth performance of corals can be influenced by several physical, chemical and biological parameters. For corals harbouring zooxanthellae, light is one of such key factors, as it can influence the photosynthetic performance of these endosymbionts, as well as coral physiology, survival and growth. The economic feasibility of ex situ coral aquaculture is strongly dependent on production costs, namely those associated with the energetic needs directly resulting from the use of artificial lighting systems. In the present study we developed a versatile modular culture system for experimental coral production ex situ, assembled solely using materials and equipment readily available from suppliers all over the world; this approach allows researchers from different institutions to perform truly replicated experimental set-ups, with the possibility to directly compare experimental results. Afterwards, we aimed to evaluate the effect of contrasting Photosynthetically Active Radiation (PAR) levels, and light spectra emission on zooxanthellae photochemical performance, through the evaluation of the maximum quantum yield of PSII (Fv/Fm) (monitored non-invasively and non-destructively through Pulse Amplitude Modulation fluorometry, PAM), chlorophyll a content (also determined non-destructively by using the spectral reflectance index Normalized Difference Vegetation Index, NDVI), photosynthetic and accessory pigments, number of zooxanthellae, coral survival and growth. We studied two soft coral species, Sarcophyton cf. glaucum and Sinularia flexibilis, as they are good representatives of two of the most specious genera in family Alcyoniidae, which include several species with interest for biotechnological applications, as well as for the marine aquarium trade; we also studied two commercially important scleractinian corals: Acropora formosa and Stylophora pistillata. We used different light sources: hydrargyrum quartz iodide (HQI) lamps with different light color temperatures, T5 fluorescent lamps, Light Emitting Plasma (LEP) and Light Emitting Diode (LED). The results achieved revealed that keeping S. flexibilis fragments under the same light conditions as their mother colonies seems to be photobiologically acceptable for a short-term husbandry, notwithstanding the fact that they can be successfully stocked at lower PAR intensities. We also proved that low PAR intensities are suitable to support the ex situ culture S. cf. glaucum in captivity at lower production costs, since the survival recorded during the experiment was 100%, the physiological wellness of coral fragments was evidenced, and we did not detect significant differences in coral growth. Finally, we concluded that blue light sources, such as LED lighting, allow a higher growth for A. formosa and S. pistillata, and promote significant differences on microstructure organization and macrostructure morphometry in coral skeletons; these findings may have potential applications as bone graft substitutes for veterinary and/or other medical uses. Thus, LED technology seems to be a promising option for scleractinian corals aquaculture ex situ.

Ex Situ Scanning Electrochemical Microscopy (SECM) Investigation of Bismuth- and Bismuth/Lead Alloy Film-Modified Gold Electrodes in Alkaline Medium

Scanning electrochemical microscopy (SECM) in feedback mode was employed to characterise the reactivity and microscopic peculiarities of bismuth and bismuth/lead alloys plated onto gold disk substrates in 0.1 molL(-1) NaOH solutions. Methyl viologen was used as redox mediator, while a platinum microelectrode was employed as the SECM tip. The metal films were electrodeposited ex situ from NaOH solutions containing either bismuth ions only or both bismuth and lead ions. Approach curves and SECM images indicated that the metal films were conductive and locally reactive with oxygen to provide Bi(3+) and Pb(2+) ions. The occurrence of the latter chemical reactions was verified by local anodic stripping voltammetry (ASV) at the substrate solution interface by using a mercury-coated platinum SECM tip. The latter types of measurements allowed also verifying that lead was not uniformly distributed onto the bismuth film electrode substrate. These findings were confirmed by scanning electron microscopy images. The surface heterogeneity produced during the metal deposition process, however, did not affect the analytical performance of the bismuth coated gold electrode in anodic stripping voltammetry for the determination of lead in alkaline media, even in aerated aqueous solutions. Under the latter conditions, stripping peak currents proportional to lead concentration with a satisfactory reproducibility (within 5% RSD) were obtained.

Vitrification with DAP 213 and Cryotop of Ex Situ and In Situ Feline Cumulus-Oocyte Complexes

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulusoocyte complexes (COCs) vitrified with DAP 213 (2 M DMSO, 1 M acetamide, 3 M propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm3) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29-40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.

The use of vinasse as an amendment to ex-situ bioremediation of soil and groundwater contaminated with diesel oil

Este trabalho investigou a possibilidade de se usar a vinhaça como um agente estimulador de processos de biorremediação ex-situ. Amostras de água subterrânea e solo foram coletadas em três postos de combustíveis. A biorremediação do solo foi simulada em frascos de Bartha, usados para medir a produção de CO2, durante 48 dias, onde a vinhaça foi adicionada a uma concentração de 33 mL.Kg-1 de solo. A eficiência de biodegradação também foi medida pela quantificação de hidrocarbonetos totais de petróleo (TPH) por cromatografia gasosa. A biorremediação da água subterrânea foi realizada em experimentos laboratoriais simulando condições aeradas (bioreatores) e não aeradas (frascos de DBO). em ambos os casos, a concentração de vinhaça foi de 5 % (v/v) e diferentes parâmetros físico-químicos foram avaliados durante 20 dias. Embora um aumento da fertilização e da população microbiana do solo foram obtidos com a vinhaça, esta estratégia não se mostrou adequada em aumentar a eficiência da biorremediação dos solos contaminados com óleo diesel. A adição de vinhaça às águas subterrâneas contaminadas teve efeitos negativos na biodegradação dos hidrocarbonetos, uma vez que a vinhaça, como uma fonte de carbono facilmente assimilável, foi preferencialmente consumida.

Diversidade genética e tamanho efetivo de duas populações de Myracrodruon urundeuva Fr. All., sob conservação ex situ

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biotecnologia para conservação ex-situ de plantas medicinais do Cerrado

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Conservação genética Ex Situ de populações naturais de Myracrodruon urundeuva Fr. All. em sistema silvipastoril

Pós-graduação em Agronomia - FEIS

EX-SITU BIOREMEDIATION OF BRAZILIAN SOIL CONTAMINATED WITH PLASTICIZERS PROCESS WASTES

The aim of this research was to evaluate the bioremediation of a soil contaminated with wastes from a plasticizers industry, located in Sao Paulo, Brazil. A 100-kg soil sample containing alcohols, adipates and phthalates was treated in an aerobic slurry-phase reactor using indigenous and acclimated microorganisms from the sludge of a wastewater treatment plant of the plasticizers industry (11gVSS kg(-1) dry soil), during 120 days. The soil pH and temperature were not corrected during bioremediation; soil humidity was corrected weekly to maintain 40%. The biodegradation of the pollutants followed first-order kinetics; the removal efficiencies were above 61% and, among the analyzed plasticizers, adipate was removed to below the detection limit. Biological molecular analysis during bioremediation revealed a significant change in the dominant populations initially present in the reactor.

Batrachochytrium dendrobatidis in amphibians confiscated from illegal wildlife trade and used in an ex situ breeding program in Brazil

This paper describes an outbreak of chytridiomycosis affecting a group of Dendrobates tinctorius, a Neotropical anuran species, confiscated from the illegal wildlife trade and housed in a private zoo in Brazil as part of an ex situ breeding program. We examined histological sections of the skin of 30 D. tinctorius and 20 Adelphobates galactonotus individuals. Twenty D. tinctorius (66.7%) and none of the A. galactonotus were positive for Batrachochytrium dendrobatidis (Bd). Multiple development stages of Bd infection were observed. The reasons for the interspecific difference in the rate of infection could not be determined, and further studies are advised. Because the examined population consisted of confiscated frogs, detailed epidemiological aspects could not be investigated, and the source of the fungus remains uncertain. The existence of ex situ amphibian populations is important for protecting species at higher risk in the wild, and ex situ amphibian conservation and breeding programs in Brazil may be established using confiscated frogs as founders. However, this paper alerts these programs to the urgency of strict quarantine procedures to prevent the introduction of potential pathogens, particularly Bd, into ex situ conservation programs.

Genetic diversity and population structure of Musa accessions in ex situ conservation

Abstract Background Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa. Results From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis. Conclusions The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.

Ex-situ bioremediation of Brazilian soil contaminated with plasticizers process wastes

The aim of this research was to evaluate the bioremediation of a soil contaminated with wastes from a plasticizers industry, located in São Paulo, Brazil. A 100-kg soil sample containing alcohols, adipates and phthalates was treated in an aerobic slurry-phase reactor using indigenous and acclimated microorganisms from the sludge of a wastewater treatment plant of the plasticizers industry (11gVSS kg-1 dry soil), during 120 days. The soil pH and temperature were not corrected during bioremediation; soil humidity was corrected weekly to maintain 40%. The biodegradation of the pollutants followed first-order kinetics; the removal efficiencies were above 61% and, among the analyzed plasticizers, adipate was removed to below the detection limit. Biological molecular analysis during bioremediation revealed a significant change in the dominant populations initially present in the reactor.

Conservación ex situ de Cymodocea nodosa (Ucria) Ascherson. Propagación de semillas in vitro, y establecimiento de cultivos celulares.

[ES] El objetivo principal de esta tesis ha sido presentar soluciones a los problemas de conservación que afectan a la especie Cymodocea nodosa y su ecosistema en Canarias, llevando a cabo estrategias de conservación ex situ que incluyen el desarrollo de una técnica de propagación con la que obtener plántulas germinadas a partir de semillas, su aclimatación y trasplante al mar. Una vez desarrollada la técnica de propagación in vitro, nos planteamos abordar estrategias para optimizar el procedimiento establecido, interviniendo sobre la conservación de las semillas in vitro y sobre la mejora, previo al trasplante, del estatus nutritivo y del crecimiento de las plántulas. Con la experiencia adquirida se han establecido las bases del cultivo in vitro a partir de tejido embrionario, permitiendo el mantenimiento de cultivos celulares en condiciones asépticas. El protocolo desarrollado supone una herramienta nueva y fundamental para el futuro del cultivo in vitro de esta especie, ya que permite ensayar nuevas técnicas y protocolos de regeneración para poder obtener, en un futuro, embriones somáticos que se desarrollen hasta plantas completas aplicando la técnica de germinación y propagación desarrollada, y proveer, así, de material vegetal para llevar a cabo programas de restauración y trasplante sin recurrir a las praderas naturales.

Ex situ X-­Ray absorption study of Li-­rich layered cathode material Li[Li0.2Mn0.56Ni0.16Co0.08]O2

The Li-rich layered transition metal oxides (LLOs) Li2MnO3-LiMO2 (M=Mn, Co, Ni, etc.) have drawn considerable attention as cathode materials for rechargeable lithium batteries. They generate large reversible capacities but the fundamental reaction mechanism and structural perturbations during cycling remain controversial. In the present thesis, ex situ X-ray absorption spectroscopy (XAS) measurements were performed on Li[Li0.2Mn0.56Ni0.16Co0.08]O2 at different stage of charge during electrochemical oxidation/reduction. K-edge spectra of Co, Mn and Ni were recorded through a voltage range of 3.7-4.8V vs. Li/Li+, which consist of X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). Oxidation states during initial charge were discussed based on values from literature as well as XANES analysis. Information about bond distance, coordination number as well as corresponding Debye-Waller factor were extracted from Gnxas analysis of raw data in the EXAFS region. The possibility of oxygen participation in the initial charge was discussed. Co and Ni prove to take part in the oxidation/reduction process while Mn remain in the tetravalent state. The cathode material appears to retain good structural short-range order during charge-discharge. A resemblance of the pristine sample and sample 4 was discovered which was firstly reported for similar compounds.