Dissertations / Theses on the topic 'Gracilaria gracili'

Includes bibliographical references.<br>In the past, commercial quantities of G. gracilis were harvested from Saldanha Bay until population collapses of the natural resource necessitated the need for research into alternative aquaculture programs for G. gracilis cultivation. One of the reasons for the G. gracilis population collapse was attributed to adverse conditions during summer which led to thermal stratification of the water column and subsequent nutrient limiting conditions. Inorganic nitrogen has been identified as the major nutrient factor limiting growth and production of G. gracilis populations in South Africa. Although the physiological mechanisms implemented by G. gracilis which allow adaption to low nitrogen environments have been investigated, not much is known about the molecular mechanisms which underlie these adaptions. Thus, it is necessary to elucidate the molecular basis of these adaptions in G. gracilis to complement the existing physiological data.

Cette these a consiste en une etude ecologique de deux especes de rhodophycees (gracilaria bursa-pastoris et gracilaria gracilis) dans l'etang de thau (herault, france). L'etude a consiste en suivi des population naturelles : analyse de la biomasse, de la composition pigmentaire, du taux de croissance, de la reproduction, de la composition pigmentaire de la production et de la qualite de l'agar en fonction des parametres environnementaux (temperature, salinite, lumiere, sels nutritifs, etc. ). Cette etude a montre des differences entre les deux especes avec des periodes de developpement et de reproduction bien distincts. Il existe une dominance de g. Bursa-pastoris sur g. Gracilis. Cette suprematie est illustree par une biomasse plus importante, deux periodes de croissance, la presence de plants fertiles durant toute l'annee et une grande capacite photoadaptative. Les deux especes ont aussi montre des differences quant a la production et a la qualite de l'agar. Ces deux parametres sont influences par les conditions du milieu (temperature, salinite), l'etat nutritionnel des algues (contenu interne en azote) et sont directement lies a la croissance. Les deux gracilaires presentent de fortes capacites d'adaptation au milieu lagunaire, resultats de strategies ecologiques particulieres (eurythermie, reproduction, photoadaptation, etc. ), qui expliquent leur abondance et leur perennite dans l'etang de thau.

Bibliography: leaves 86-97.<br>Low environmental nutrient concentration is the main factor limiting natural production and success of Gracilaria gracilis cultivation in Saldanha Bay, South Africa and nitrogen is the single element of all the nutrients required by seaweeds that is most frequently limiting to growth. Biomass, relative growth rate, the concentration of nitrogen in the growth medium, the mean nitrogen uptake rate of the plant and the amount of nitrogen in the thallus were determined for nitrogen enriched and nitrogen deprived G. gracilis cultures grown in the laboratory.

Includes bibliographical references.<br>The red agarophytic alga, Gracilaria gracilis, occurs naturally within Saldahna Bay, South Africa. Gracilaria species are commercially exploited for their hydrocolloid agar, valued at US$132 million per annum (FAO, 2002). G. gracilis is thus a valuable resource for South Africa in terms of generating foreign currency through export. Nitrogen limitation occurring within Saldahna Bay during the summer to autumn months, however, is considered to be the major factor preventing commercial cultivation of G. gracilis. Algae possess various survival mechanisms that generally involve the activation or repression of various gene regulatory systems. Such regulatory systems aid in preserving energetic homeostasis at a cellular level, thereby allowing maximal algal survival during adverse environmental conditions. Microarray technology is a highthroughput global gene analysis tool, allowing for the simultaneous identification of thousands of differentially expressed genes in a single assay. Combined with genomic technologies such as genetic engineering, microarray technology has the potential to identify genes that confer enhanced in vivo tolerance to a specific stress. In the current investigation, microarray technology was utilized to identify genes differentially expressed in G. gracilis in response to nitrogen limitation. A total of 39 differentially expressed genes were identified. Ofthe 35 genes that were sequenced, 13 were assigned a putative function based on sequence similarity to genes deposited in various databases. The remaining sequences corresponded to either unknown hypothetical proteins or else had no significant homology to any of the sequences in the databases. Overall, the current investigation has provided a foundation for future research into the biological role(s) of each of the identified differentially expressed genes within their true isogenic background. Future studies may thus allow for improved crop tolerance and stability, enhancing aquaculture of this valuable resource in Southern Africa.

Bibliography: pages 132-157.<br>The growth of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham was examined by studying the effect of organismic determinants such as thallus length, position along the thallus and branching in a series of in situ and laboratory-based experiments. Knowledge of these factors is essential in order to maximise production from suspended seaweed rafts seeded with vegetative G. gracilis fragments. Seeding netlons with freshly collected material provided up to 30 % higher relative growth rates than seaweed maintained on the netlons for three successive months. Initial seedstock length greatly affected growth rate and yield such that 30 cm thalli fragments resulted in growth rates 14 % higher than for 10 cm fragments. This difference is suggested to be due to higher contribution of growth by lateral branches to overall biomass. Comparisons of the growth of apical and basal fragments suggested that growth takes place over the entire length of the thallus but that the apex contributes more to overall elongation than does the proximal part. The removal of apical meristems resulted in an enhanced branching frequency with production of four times as many branches as intact fragments. Evidence is also provided for severe morphological differentiation following long periods of rapid growth. These thalli have very high frequency of branching, are hollow due to the disintegration of medullary cells and are considered to be completely senescent.

Gracilaria gracilis occurs naturally in Saldanha Bay, and was important to the South African export industry as it is a source of two important types of agar, namely food grade and sugar reactive agar. However, a number of abiotic and biotic factors, such as nutrient limitation, has virtually destroyed the G. gracilis resource. An understanding of the physiological response of the alga to nutrient limitation will aid in re-establishing and sustaining G. gracilis populations. However, modelling algal physiology and growth in response to abiotic stresses such as nutrient limitation requires an understanding of the underlying metabolic processes. The present study aimed to address this by investigating nitrogen metabolism and the mechanisms regulating nitrogen metabolism in G. gracilis. This was achieved by profiling changes in gene and protein expression, and activity of two major nitrogen metabolic enzymes, nitrate reductase and glutamine synthetase. Long term culture of G. gracilis in nitrogen replete and lacking conditions indicated that nutrient limitation causes a reduction in intracellular nitrogen and nitrogen protein stores such as phycoerythrin. When various sources were introduced to the culture medium to replenish nitrogen starved G. gracilis, changes in nitrate reductase and glutamine synthetase mRNA, protein and activity seemed to be dependent on the nutrient history of the cells, intracellular and extracellular nitrogen concentrations, metabolites of nitrogen assimilation and other metabolic processes such as carbon metabolism and photosynthesis. Nutrient studies suggested that multiple G. gracilis nitrate reductase and glutamine synthetase isoforms are present and differentially regulated via transcriptional, post-trancriptional, translational and posttranslational mechanisms. Furthermore, the insensitivity of these nitrogen metabolic enzymes to ammonium inhibition and the ability to alter the GS1:GS2 activity ratio possibly represents adaptive strategies developed by G. gracilis to survive nitrogen limitation. Immunocytochemical investigations confirmed the presence of multiple nitrate reductase and glutamine synthetase isoforms. The enzymes were successfully localised to the cell wall, chloroplast and cytosol of G. gracilis. A novel finding was the immuno-localisation of glutamine synthetase to intracellular starch granules. Overall, findings in the current study have suggested multiple roles for these metabolic enzymes that include nitrogen assimilation/transport, cell wall biosynthesis and senescence. This study led to the development of a model of the metabolic changes that occur in nitrogen replete and deplete G. gracilis and provides a firm foundation for future studies of the nitrogen stress response in G. gracilis. Characterisation of the G. gracilis nitrogen stress response may ultimately revive mariculture of this commercially important alga in South Africa.

The addition of a local commercial seaweed extract (Kelpak®) to crop plants has proven to be beneficial as it improves growth and yields. Its efficiency has been attributed to its production method that involves a cold process, resulting in a product containing significant amounts of plant growth regulators (auxins and cytokinins). The aim of this study was therefore to investigate the effects of this commercial seaweed concentrate (Kelpak®) on the growth of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham, with a view to the potential in mariculture, especially as this red seaweed is currently under cultivation in South Africa as feed in abalone aquaculture.

Bibliography: leaves 190-209.<br>The red agar-containing macroalga G. gracilis (Stackhouse) Steentoft, Irvine et, Farnham has occurred in Saldanha Bay, South Africa for many years. However, in recent years a number of collapses in this G. gracilis population were recorded, in some instances almost erradicating the entire population. One of the causes of these collapses is thought to be bacterial disease about which there is very little known. The bacterial pathogens of this macroalga were thus investigated to determine the nature of disease occurrence and how G. gracilis responds to such infections. A large number of culturable bacterial epiphytes isolated from G. gracilis from Saldanha Bay, South Africa, and Luderitz, Namibia were characterised and compared. The number of culturable bacteria isolated from the seawater surrounding the macroalgae was significantly lower than that which occurred epiphytically on the macro algal thalli. Most of the bacteria isolated were Gram-negative, motile rods, and many were classified to genus level. Scanning electron microscopy confirmed that a large epiphytic population of coccoid and rod-shaped bacteria occurs primarily on the main thalli of the rnacroalga and that significantly fewer (and often none) reside on the thallus growth tips.

Includes bibliographical references.<br>Gracilaria gracilis is a commercially important alga, previously harvested from the wild South African population in Saldanha Bay as a feed for marine organisms and as a source of commercially important agar. Since 1974 however, a number of sporadic population collapses has lead to the destruction of this once flourishing resource. After numerous failed attempts at re-establishing this industry, the need to develop an alternative farming strategy became evident. In order to devise such a solution, a better understanding of the tolerances and responses of this alga to the environmental parameters responsible for the downfall of the population is required. Although the exact reasons remain unclear, Jaffray et al., 1997 have reported that increased water temperature in Saldanha Bay may be a contributing factor as the population collapses have repeatedly occurred during summer months. Thus the effect of heat stress on G. gracilis has been selected for this study.

Includes abstract.<br>Includes bibliographical references.<br>A better understanding of how this commercially important seaweed responds at a genetic level to stresses faced in the aquaculture environment would not only be advantageous to the South African industry, but this knowledge is essential for selecting and/or engineering macroalgal strains that are either more tolerant or resistant to these stresses. This requires in vivo analysis of G. gracilis gene function and regulation in order to introduce new or improved genes into G. gracilis, and for this to be possible, a protocol for transformation of recombinant DNA into G. gracilis is required. In this study a transformation and tissue culture system for G. gracilis was developed. These tools provide the necessary groundwork for future genetic manipulation studies that are essential for improving our understanding of the role that various genes play in stress response and tolerance in G. gracilis.

Les algues rouges a cycle de vie haplo-diploide isomorphe ont des caracteristiques particulieres : (1) trois types d'individus macroscopiques coexistent dans les populations : tetrasporophytes diploides, gametophytes haploides males et femelles ; (2) trois types de propagules assurent la dispersion : spores haploides, spores diploides et gametes males ; (3) les gametes sont produits par mitoses sur les individus haploides ; et (4) chaque fecondation est suivie d'une multiplication du zygote par mitoses donnant des milliers de spores diploides genetiquement identiques. Cette etude explore l'impact des ces differentes caracteristiques sur les fonctionnements demographique et genetique des populations de gracilaria gracilis du detroit du pas-de-calais a l'aide de trois approches : demographie, genetique des populations et analyse de paternite. L'etude demographique montre que les parametres demographiques varient peu dans l'espace et dans le temps. Les populations montrent un taux de croissance modeste, un temps de generation long ; les taux de survie haploide et diploide sont eleves tandis que leurs taux de recrutement sont faibles. L'etude de la structure genetique montre que les flux geniques entre populations sont importants (a l'echelle de 5 km), mais qu'une differenciation genetique peut s'etablir entre populations selon leur position sur l'estran, independamment de la distance geographique qui les separe. Les analyses de paternite montrent que l'essentiel des fecondations se produit dans un rayon d'un metre, mais que la migration des gametes males entre populations est non negligeable. De plus, les descendances sont engendrees par une multitude de males et il semble qu'il y ait une competition entre males pour acceder aux gametes femelles. Ces resultats depeignent une image du fonctionnement des populations de gracilaires : stables, renfermant une forte diversite genetique et ou la selection sexuelle est potentiellement une force evolutive.

For suspended cultivation of seaweeds in the open ocean, the selection of suitable sites and the optimization of cultivation methods for maximum productivity are of utmost importance. Growth of raft-cultivated Gracilaria gracilis in Saldanha Bay is known to be badly affected in late summer due to the persistence of warm oligotrophic water at the ocean surface which prevents replenishment of nutrients by upwelling. This study documents the extent of nitrogen starvation during this period and investigates some possible methods of managing this problem. Nitrogen content, C:N ratio and stable isotope abundances of C and N were obtained for material grown for varying lengths of time, material grown at various depths and material that had been fertilized prior to placement on the raft. The relationship between thallus colour and nitrogen content was also documented using a standard colour guide. The onset of nitrogen starvation was rapid, occurring after about two weeks on the raft. Growth was optimized about a meter below the water surface, possibly as a result of photoinhibition of photosynthesis at the surface. Nitrogen limitation ceased below about 2m growth depth. Prefertilization had no significant effect on yield after 4 weeks growth. Thallus colour showed a clear relationship with N content and may be a useful indicator of N status of cultivated Gracilaria. δ¹⁵N was related to N content and increased rapidly when N limitation was removed. Stable isotope natural abundance measurements are a useful tool in the study of growth environment, but more controlled experimentation investigating the factors which influence these values is needed, particularly for macroalgae.

To our knowledge, this study represents the first analysis of gene expression using cDNA microarrays in the red macroalga G. gracilis. Western hybridization analysis was used to establish whether the observed changes in gene expression following exposure to disease elicitors positively correlated to changes at the protein level.

Rising temperature caused by global warming alters physiology, phenology and/or distribution in a wide array of plant and animal species, which has dramatic knock-on effects at different levels of organisation. This study investigates the individual and interactive effects of temperature (18ᵒC, 22ᵒC and 30ᵒC) and additions of the macroalga Gracilaria gracilis (high and low) on the performance of the seagrass Zostera capensis, which occurs in Langebaan Lagoon, South Africa over a seven-week period. Results from the laboratory experiment revealed that G. gracilis did not significantly affect the performance of Z. capensis although temperature did result in greater leaf width, fouling and senescent biomass, as well as marginally greater leaf area and lower below-ground biomass at 30ᵒC. Increasing temperature also increased G. gracilis biomass, percent cover and fouling by microalgae. In addition, there was no interaction between temperature and the additions of Gracilaria. The overall findings of this study indicate that Z. capensis abundance is likely to decrease while G. gracilis will conversely increase in abundance in response to warming. Changes in abundance of those two ecosystem engineers highlight the possibility of a phase shift from a seagrass- to macroalgal-dominated state in Langebaan Lagoon.

Cette these retrace les differentes etapes de la recherche et de la caracterisation de loci microsatellites chez gracilaria gracilis, une algue rouge haplo-diploide. Elle presente egalement les premiers resultats obtenus sur l'analyse de la structuration genetique des populations. La recherche de marqueurs se s'est averee tres difficile et la construction et l'analyse de deux banques genomiques n'ont permis que l'identification de deux loci polymorphes, gv1ct et gv2ct. Pour gv1ct, la determination de conditions de typages a elle-meme ete tres delicate. Cependant, les resultats obtenus nous permettent aujourd'hui de proposer un certain nombre d'ameliorations pour optimiser cette phase. Les premiers resultats sur le fonctionnement des populations mettent en evidence des differenciations genetiques entre populations et un isolement par la distance. Ils permettent egalement d'acceder aux flux geniques intra-population par une analyse de paternite. Ces resultats sont par ailleurs en contradiction avec nos hypotheses de depart sur le regime de reproduction, car ils montrent que cette espece est plutot de type allogame. Par ailleurs, ce travail a permis de mettre en avant un certain nombre de limites dans l'utilisation des marqueurs microsatellites. En effet, le locus gv1ct est caracterise par un niveau de polymorphisme associe a une gamme de variation de taille extraordinaire entrainant des difficultes d'amplification des grands alleles. Ces difficultes ont conduit a l'observation, dans certaines conditions defavorables de typages, d'alleles nuls chez les heterozygotes (dominance du petit allele). Enfin, le locus gv2ct nous a permis d'aborder l'evolution moleculaire des microsatellites. En effet, le sequencage des alleles au locus gv2ct revele l'existence : (i) d'une homoplasie de taille et (ii) de pas de mutation pouvant etre complexes ne faisant pas intervenir de simples variations du nombre de repetitions de l'unite principale du microsatellite.

Mastocarpus stellatus et Gracilaria gracilis sont des algues rouges présentes à la fois sur les côtes atlantiques françaises et vietnamiennes. Elles constituent une biomasse disponible et valorisable au travers de leur pigment la R-PE. L’objectif de ce travail est d’une part d’acquérir des connaissances sur la composition biochimique de ces macroalgues et d’autre part d’optimiser les paramètres d’extraction de la R-PE à partir de ces deux modèles d’étude. Ainsi, deux méthodes d’extraction ont été mises en œuvre: la macération classique (eau, tampon) et l’extraction assistée par enzyme (cellulase, xylanase, B-glucanase). Le prétraitement de l’algue a aussi été étudié. Les meilleurs rendements d’extraction de la R-PE ont été obtenus à partir des algues lyophilisées et cryobroyées en mettant en œuvre le génie enzymatique. L’utilisation des plans d’expérience utilisant la méthodologie des surfaces de réponse ont permis d’optimiser les conditions d’hydrolyse (température, pH, rapport E/S). L’enzyme ainsi que les conditions d’hydrolyse différent selon l’espèce. Parallèlement, un protocole de purification par chromatographie échangeuse d’anions a permis d’obtenir un pigment concentré et purifié (IP = 3,25) à partir d’un extrait de Gracilaria gracilis<br>Mastocarpus stellatus and Gracilaria gracilis are endemic red seaweeds commonly funded in French Atlantic and the coast of Vietnam. They represent an available and usable biomass through the extraction of their original proteinic pigment R-Phycoerythrin. The purpose of this work was to increase the scientific knowledge on the biochemical characterization of these two species on one side. On the other side, this work dealt with the optimization of the extraction parameters of the R-PE. Hence, two extraction procedures have been compared: conventional maceration (with investigation of extracting solution) and enzymatic-assisted extraction (cellulase, xylanase, B-glucanase). Algal prior treatment has also been studied. Highest R-PE extraction yields have been obtained when enzymatic-assisted extraction had been employed on cryo-ground seaweeds. The use of response surface methodology analysis allowed to improve simultaneously the hydrolysis conditions (temperature, pH, E/S ratio) and the purity and extraction yields of R-PE. Finally, a one-step chromatographic method by anion-exchange chromatography had been developed to obtain a concentrated and purified (PI = 3.25) pigment from Gracilaria gracilis

Includes bibliographical references (leaves 91-111).<br>The feasibility of growing red seaweed, Gracilaria gracilis (Gigartinales, Rhodophyta) in land-based culture systems was investigated in two Western Cape abalone farms i.e. Jacobsbaai Sea Products (JSP) and Irvin & Johnson (I&J) and large size culture tanks (I&J). All the tanks were stocked with at 2 kg m⁻² supplied with unfiltered seawater (24 and 4 volume exchanges d⁻¹) JSP and I&J, respectively. This study also tested the efficiency of G. gracilis as a biofiltering species as well as investigating the effectivenss and influence of nutrient-enriched seawater i.e. turbot and abalone effluent (JSP) and fertilized seawater and abalone effluent (I&J) on the growht of Gracilaria.

Includes abstract<br>Includes bibliographical references<br>Gracilaria gracilis that grows naturally at Saldanha Bay, South Africa is economically important as a source of agar. The Gracilaria yields from natural beds at Saldanha Bay are however unreliable, and consequently the South African Gracilaria industry has experienced a number of setbacks over the years. The only way a consistent supply can be assured is by mariculture to supplement the natural harvests. In 1993 the Seaweed Research Institute (SRU) found that mariculture of G. gracilis in Saldanha Bay is feasible but that there is potential to improve yields by technical research and development (Anderson et al.1996a). Jaffray and Coyne (1996) developed a pathogenicity assay that demonstrated that agarolytic bacteria isolated from Saldanha Bay Gracilaria induced disease symptoms such as thallus bleaching, while non-agarolytic isolates did not. It is thought that unfavorable environmental conditions such as elevated water temperature and nutrient depletion, which occur during the summer months in the surface layers of the water column in Saldanha Bay, induce the onset of agarase production or result in changes in the bacterial community structure in which agarase-producers become more dominant. By using the pathogenicity assay, Jaffray and Coyne (1996) identified the highly agarolytic Gracilaria gracilis pathogen, Pseudoalteromonas sp. LS2i. The aim of this study was to characterize the bacterial pathogen, Pseudoalteromonas sp. LS2i to further our understanding of virulence regulation and specifically, the role of the agarase enzymes in the process of seaweed-pathogen interaction. Two agarolytic clones, pEB1 and pJB1, were obtained after constructing and screening a Pseudoalteromonas sp. LS2i genomic library in Esherichia coli. Restriction enzyme mapping suggested that both clones contain the same agarase gene. Southern hybridization studies confirmed the origin of the cloned DNA and sequencing studies revealed the 1062 bp ORF, putative promoter region, putative ribosome binding site and putative transcriptional start point of the cloned agarase gene. The ORF showed sequence identity to several other β-agarases and was identified as a member of the GH-16 family of glycoside hydrolases. The agarase was purified from the E. coli JM109 (pEB3) transformant. The molecular weight was estimated to be 39 kDa by SDS-PAGE. Zymogram analysis confirmed that the purified protein is agarolytic and TLC analysis revealed that the predominant end-products of agar hydrolysis are neoagarohexaose and neoagarobiose, which indicates the same mode of action as that observed for the agarase produced extracellularly by Pseudoalteromonas sp. LS2i.

Os corantes alimentares sintéticos têm sido utilizados na indústria alimentar durante séculos, mas hoje em dia com a mudança das tendências de consumo, existe uma grande procura por alternativas naturais com capacidade de substituir esses corantes. As ficobiliproteinas (PBPs) são proteínas solúveis em água presentes maioritariamente nas algas vermelhas e cianobactérias, com grande potencial económico nas indústrias farmacêuticas, biomédicas, alimentares e cosméticas. Estas proteínas podem ser classificadas em três grupos principais, baseados no seu comportamento de absorção: Ficoeritrinas (PE), Ficocianinas (PC) e Aloficocianinas (APC) com máximos de absorção entre os 540-570, 610-620 e 650-655 nm, respetivamente. Este trabalho tem como objetivo principal otimizar as condições de extração das PEs a partir da alga vermelha Gracilaria gracilis, presente na costa portuguesa que contém PBPs, principalmente PEs, como pigmentos acessórios, sendo uma alga principalmente utilizada como alimento e fonte de agar. Adicionalmente, foi estudado o efeito do pH e da temperatura na estabilidade dos extratos aquosos, bem como a sua aplicação em produtos alimentares. Foram estudadas cinco metodologias distintas, com elevado potencial em termos económicos e ambientais: maceração com almofariz, banho de ultrassons, sonicador, congelamento/descongelamento e altas pressões. As condições experimentais, tais como a concentração do tampão fosfato (C), o rácio solvente/biomassa (R), o tempo de homogeneização (t1), o tempo de extração (t2) e a pressão (P) foram otimizadas utilizando a Metodologia de Superfície de Resposta (RSM). Esta permitiu delinear modelos sustentados nas variáveis independentes e explicar a variabilidade que ocorre nos parâmetros considerados significativos (rendimento). A maceração foi o método de extração mais eficiente (condições ótimas t1 = t2 = 10 min; C = 0,1 M; R = 1:50) atingindo valores de 3,583 ± 0,033 mg de PE/g de alga rendimentos 55-65 % superiores aos outros métodos testados. Quanto às condições, a variável t2 foi a que exerceu maior influência no rendimento obtido, enquanto o aumento de C resultou em menores rendimentos. t1 foi a avariável que menos efeito teve no resultado. Relativamente à estabilidade, foi monitorizado o efeito de diferentes temperaturas de armazenamento (-20, 4, temperatura ambiente (~19) e 40 ºC) e pH (4, 6,9 e 8) na concentração de PE durante 10 dias. Os resultados demonstram maior estabilidade a -20 ºC e pH 6,9. Como caso de estudo, o extrato purificado (pigmento) foi incorporado em panquecas (0,15 %) e iogurtes (0,12 %) como corante rosa. As PEs foram precipitadas com sulfato de amónio (65 %) e purificadas por diálise, permitindo a utilização em alimentos. Durante o processo de cozimento das panquecas, foi observada uma ligeira perda de cor, enquanto nos iogurtes a cor manteve-se praticamente inalterada durante mais de dois meses, sugerindo uma melhor aptidão deste tipo de produto para a coloração com PEs. Com este trabalho foi possível concluir que a Gracilaria gracilis tem potencial enquanto fonte de pigmentos para aplicações alimentares, especialmente para utilização em produtos que não necessitem de elevadas temperaturas no processo de produção.

Con le crescenti pressioni sugli stock ittici naturali determinate dai fenomeni sempre più preoccupanti di sovrapesca, l’acquicoltura rappresenta il futuro della produzione di specie ittiche destinate al consumo umano. Questo settore presenta diversi problemi in termini di impatto ambientale, fra cui i più importanti risultano essere legati al sovraccarico di nutrienti nelle acque di scarico degli impianti. Purtroppo però, ad oggi, non esistono realtà consolidate (almeno nell’acquicoltura italiana) che prendano in seria considerazione il problema della sostenibilità ambientale delle pratiche acquicolturali. Una presa di coscienza, però, sarà necessaria a breve, soprattutto considerando le sempre più restrittive leggi che regolano l’immissione di reflui nei corpi idrici naturali. La problematica quindi, va affrontata in maniera metodica e con soluzioni che possano rappresentare un ricavo e non un costo, in modo da invogliare gli imprenditori del campo acquicolturale a investire in tali espedienti; questi potenzialmente potrebbero contribuire a ridurre notevolmente il rischio di eutrofizzazione. Con il presente lavoro, argomentato nei suoi quattro obiettivi realizzativi, si è voluto creare un protocollo da poter applicare e sfruttare nel contesto acquicolturale nazionale. Infatti, nell’OR1 si è identificato l’organismo (G. gracilis) che potesse avere le caratteristiche di: adattabilità alla coltura in ambiente naturale e in impianto, alto valore commerciale, e buon potere “depurante”. Per cui, nell’OR2 si è stabilito quale fosse il metodo migliore per l’accrescimento di questa specie in modo da poterlo applicare e sperimentare, nell’OR3, nelle acque di scarico di un impianto di acquicoltura. Infine tramite l’OR4 si sono stabilite le ulteriori potenzialità biotecnologiche della G. gracilis. Il presente studio ha dimostrato, infatti, come la Gracilaria sia un ottimo candidato da applicare in acquicoltura multi-trofica integrata negli impianti di acquicoltura in-shore in modo da ridurre l’eccesso di sali nutritivi disciolti nei reflui. A favore del suo utilizzo contribuiscono una serie di motivazioni: • Buone capacità adattative ad ambienti acquatici con parametri chimico-fisici anche instabili • Elevato valore economico derivante dalla possibile estrazione di agar di buona qualità (OR1) • Ottimi valori di crescita ottenuti grazie alla sperimentazione di una nuova metodologia (OR2) • Buoni risultati nell’abbattimento dei sali nutritivi nei reflui di impianti di acquicoltura (OR3) • Potenziale applicazioni biotecnologiche: o supplemento funzionale nell’alimentazione di pesci (OR4, A.4.1) o potenzialità antibatterica dei suoi estratti (OR4, A.4.2) Tenuto conto delle sopraelencate caratteristiche, la G. gracilis costituisce un organismo che potrebbe essere abbondantemente utilizzato nell’industria acquicolturale, valorizzata non solo dai possibili effetti “salutari” ma anche da un’ottima ricaduta economica.

The aim of this study was to evaluate the effect of Gracilaria gracilis inclusion in European seabass (Dicentrarchus labrax) diets and to analyse if the application of technological processes and additives in these diets can enhance their benefits to fish. The fish parameters evaluated were growth performance, feed efficiency, body composition, apparent digestibility of nutrients, innate immune response, and intestinal morphology. Six experimental diets were formulated to be isoproteic (53.4% Dry matter), isolipidic (14.6% Dry matter) and isoenergetic (22.3 kJg-1 Dry matter). A commercial-based diet was used as control diet (CTRL) and compared to other five diets with 8% inclusion of G. gracilis, namely: entire G. gracilis (GRA), after physically process (PHY), supplemented with 0.02% of phytogenic compounds (PFA), 2.5% of oligo-alginates (OLIGO), or 0.08% of nucleotides (NUCL). Dietary treatments had no effect on growth performance, voluntary feed intake, feed conversion ratio, protein efficiency, and final whole body composition. There was a decrease of muscle dry matter with the inclusion of phytogenic compounds and oligo-alginates comparing with the CTRL group. Dietary treatments with entire G. gracilis, phytogenic compounds and oligo-alginates had decreased the hepatosomatic index comparing to the CTRL group. The physical process of G. gracilis improved the apparent digestibility coefficient and nutrient balance of diets with G. gracilis inclusion. Furthermore, the oligo-alginates and nucleotides increased the protein digestibility of these algae. The physical process and oligo-alginate increased the nitrogen gain and decreased the nitrogen faecal losses. In relation to phosphorus digestibility, there was only a significantly decrease in fish fed with NUCL, which was not reflected in phosphorus faecal losses. The innate immune system was not affected by the dietary seaweed treatments. There was a decrease of villus width in anterior intestine with GRA diet, which was improved by physical process (Patent WO/2019/171293), oligo-alginates and nucleotides. There was a positive correlation between villus width and the protein and energy digestibility. Our findings suggest that physical process can improve the digestibility and nutrient absorption of diets with 8% G. gracilis without compromise growth performance, immune response and intestinal health. In addition, oligo-alginates and nucleotides could also improve G. gracilis application with advantages on protein digestibility and intestinal health.<br>Na aquacultura intensiva, a proteína representa o componente alimentar mais dispendioso. Por um longo período de tempo, a farinha de peixe tem sido a principal fonte proteica usada em peixes carnívoros devido à sua elevada quantidade proteica, digestibilidade e palatabilidade. Contudo, o limite de captura e sobreexploração de stocks selvagens de peixe restringiram a disponibilidade mundial de farinha de peixe e tornaram a sua produção insustentável, aumentando o seu preço comercial. Para além disso, a produção de farinha de peixe causa um elevado impacto ambiental devido ao combustível e energia requeridos pelos barcos de pesca, pela necessidade energética associada à produção dos pellets, e pelos efluentes ricos em partículas orgânicas. Deste modo, surgiram alternativas mais sustentáveis e menos dispendiosas como as proteínas vegetais como por exemplo cereais, soja e legumes. Porém, o elevado requerimento proteico de peixes carnívoros na sua dieta, limita a inclusão de proteínas vegetais como a principal fonte proteica. Para além disso, esta fonte pode estar associada a fatores anti nutricionais que prejudicam a sua digestibilidade, absorção de nutrientes e palatabilidade. Contudo, o aumento da procura das fontes vegetais como uma fonte proteica alternativa tem vindo a aumentar o seu preço. A ausência de água doce nem de terra arável para a sua produção, o seu elevado conteúdo proteico e perfil balanceado de aminoácidos foram algumas das vantagens para o aumento do uso de algas marinhas como uma fonte proteica alternativa na aquacultura. O género Gracilaria tem um elevado conteúdo proteico, compostos bioativos, carotenoides, atividade antiviral, antioxidante e anti-inflamatória que podem potenciar o crescimento, resposta imune e resposta intestinal dos peixes. Contudo, a sua digestão e disponibilidade de nutrientes pode ser dificultada pela presença de compostos anti nutricionais e ausência de celulases endógenas e de enzimas necessárias nos peixes carnívoros. O elevado teor de fibra nas paredes celulares das algas também pode reduzir o seu uso nas dietas de aquacultura. A elevada quantidade de fibras presentes nas paredes celulares das algas e a sua rigidez dificultam a sua digestibilidade, limitando o seu uso como fonte proteica nas aquaculturas. A biodisponibilidade, digestibilidade dos nutrientes e extração da proteína presentes nas algas pode ser potenciada pela remoção de compostos não-proteicos como a fibra. A extração da proteína é influenciada também pela elevada viscosidade e pela presença de polissacáridos, como o agar, nas paredes celulares das algas vermelhas. A eficiência da extração da proteína das algas pode ser potenciada pela rutura das paredes celulares. Para isso, processos químicos e físicos são usados nas dietas com algas para aumentar a sua digestibilidade. Processos químicos como os enzimáticos limitam a ligação entre os polissacáridos e a proteína aumentando a digestibilidade das algas. Por sua vez, os processos físicos como a moagem e o tratamento ultrassónico rompem as paredes celulares das algas melhorando a extração da proteína. Do mesmo modo, a suplementação de dietas com algas com aditivos comerciais, tais como os compostos fitogénicos, oligo-alginatos ou nucleótidos, pode melhorar a digestibilidade, palatabilidade e a resposta imune dos peixes. Os aditivos fitogénicos são compostos bioativos que derivam de plantas naturais cuja composição inclui óleos essenciais que melhoram o desempenho do peixe, a palatabilidade e o sabor da dieta. Estes atuam como suplementos naturais profiláticos que resulta numa atividade antiviral, anti microbial e anti-inflamatória, beneficiando a resposta imune. Os oligossacáridos alginatos são outro exemplo de suplementos usados na aquacultura. Estes têm uma elevada capacidade de aglutinação que reduz a lixiviação e otimiza a textura, melhorando a digestibilidade e crescimento dos peixes. Outro exemplo de suplemento são os nucleótidos, usados como uma fonte externa para potenciar processos fisiológicos como um rápido crescimento face a situações de stress. Para além disso, estes suplementos aumentam o comprimento das vilosidades intestinais, estimulam a produção enzimática, melhorando a digestão e disponibilidade de nutrientes. Deste modo, este trabalho visou avaliar o efeito da inclusão de Gracilaria gracilis em dietas de robalos (European seabass, Dicentrarchus labrax), bem como avaliar se a aplicação de processos tecnológicos e da suplementação com aditivos potenciam estas dietas. Os parâmetros avaliados foram o crescimento, eficiência alimentar, composição corporal, digestibilidade aparente dos nutrientes, resposta imune inata e da morfologia do intestino. Foram formuladas seis dietas experimentais para serem isoproteicas (53.4% Matéria seca), isolipídicas (14.6% Matéria seca) e isoenergéticas (22.3 kJg-1 Matéria seca). A dieta controlo (CTRL) foi comparada com cinco dietas teste aonde foram incluídos 8% da espécie Gracilaria, nomeadamente: alga inteira (GRA), após processo físico (PHY), suplementado com 0.02% de compostos fitogénicos (PFA), 2.5% de oligoalginatos (OLIGO), ou 0.08% de nucleótidos (NUCL). Como resultado, não houve efeitos no crescimento, consumo voluntário, índice de conversão alimentar, eficiência proteica e composição corporal do robalo alimentado com alga processada, não processada e suplementada com aditivos. A matéria seca do músculo do robalo diminuiu com a inclusão de aditivos fitogénicos e oligo-alginatos. O índice hepatosomático diminuiu significativamente nos peixes alimentados com GRA, PFA e OLIGO. O processo físico melhorou a digestibilidade aparente e o balanço de nutrientes da Gracilaria sp. A digestibilidade da proteína da G. gracilis foi potenciada com a adição de oligo-alginatos e nucleótidos. Os processos físicos e os oligo-alginatos aumentaram o ganho de nitrogénio e diminuíram as perdas fecais do mesmo. Os peixes alimentados com a dieta GRA teve maior digestibilidade do fósforo e menor perda fecal. Em relação ao sistema imune inato, não foi verificado nenhuma alteração neste parâmetro em nenhum dos tratamentos. Quanto à morfologia do intestino, a largura das vilosidades diminuiu no intestino anterior com a dieta GRA mas foram melhorados com os processos físicos e a suplementação com oligo-alginatos e nucleótidos. Houve uma correlação positiva entre a largura das vilosidades e digestibilidade da proteína e da energia. Em conclusão, apesar da inclusão de G. gracilis a 8% não ter comprometido o consumo alimentar nem o crescimento do robalo, foi verificada a sua fraca digestibilidade. Os processos físicos aplicados na dieta com algas conseguiram melhorar a sua digestibilidade que associada com o aumento do tamanho das vilosidades intestinais do intestino anterior pode promover um aumento da absorção de nutrientes, acessibilidade e digestibilidade. A digestibilidade da G. gracilis foi também melhorada com a suplementação destas dietas com oligo-alginatos e nucleótidos possivelmente devido ao aumento do tamanho das vilosidades intestinais do intestino anterior. A digestibilidade do fósforo não foi afetada pelas dietas, excepto nos peixes alimentados com nucleótidos aonde se verificou uma diminuição significativa em relação ao controlo, não se tendo refletido nas perdas fecais. Por outro lado, a inclusão de G. gracilis e de compostos fitogénicos pode aumentar a descarga de azoto no ambiente sendo uma fonte de poluição, requerendo um tratamento de efluentes.<br>This work was supported by Project “MARINALGAE4AQUA - Improving bioutilisation of marine algae as sustainable feed ingredients to increase efficiency and quality of aquaculture production”, financed by FCT-Portugal through the research project ERA-NET COFASP/004/2015