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FAIR-CT96-1518 Development of microalgal pigments for aquaculture |
Source: Final report, February, 2001.
Consortium: The project was co-ordinated by the School of Biological and Earth Sciences, Liverpool John Moores University (UK), Universidade Catolica Portuguesa, Escola Superior de Biotechnologia, Porto (Portugal), Universidad de Santiago de Compostella, (Spain), Necton Campanhia Portuguesa de Culturas Marinhas Lda, Olhao (Portugal) and the Instituto de Ciencias Biomedicas Abel Salazar, Porto (Portugal), PEP Research & Consultancy Ltd, Plymouth Univiversity, Devon (UK), Addavita Ltd., Chesterfield (UK), Sorgal, Sociedade de Oleos e Racoes SA, Porto (Portugal), Bionova, Santiago (Spain).
EXECUTIVE SUMMARY
Introduction
The global objectives of this project are:
A multidisciplinary approach was taken in an effort to ensure that both aims are fully addressed and their deliverables met.
Results
Task 1: Facilities for the laboratory-based cultivation of selected microalgae that are known accumulators of commercially important carotenoids were established by partners PI, P2, P3 and P4 in year one. Provision of facilities for the aquaculture feed trials were also established for freshwater (P6) and marine (P5) commercial fish species in year one.
A suitable strain of an astaxanthin-accumulating algal species has been selected (PI) and conditions resulting in optimal growth and carotenoid accumulation determined (PI and P3). The key parameters are nitrogen and irradiance levels. A new defined growth media specifically optimised for production of Haematococcus has been developed by P3, improving growth by approx. 5-fold. This media is available as a commercial formulation (P9). For the first time, a microplate reader (spectrophotometer) has been used to optimise conditions for algal cultivation (P3). P3 has also determined the conditions required for germination of the astaxanthin-rich cysts of Haematococcus. P3 and P9 have examined the potential use of alternative algal species, especially marine species (e.g., Nannochloropsis spp.) as a source of astaxanthin (and canthaxanthin). PI has pioneered the application of mathematical modelling of algal cultivation using 'intelligent methods'. This will allow both dynamic optimisation and process control to be applied for commercial, large-scale, production. A number of models were tested on process data for Haematococcus with very accurate performance being seen with both fuzzy-logic and, especially, neuro-fuzzy models.
Conclusions; a process for the cultivation and subsequent production of astaxanthin in Haematococcus has been defined and optimised.
Task 2: The algal astaxanthin product has been shown by both PI and P6 to be significantly more stable (both as a raw ingredient and once incorporated into feedstuffs) than the synthetic product. This appears to be due to a combination of the esterified nature of the astaxanthin found in the alga (which may effectively block the preferred sites for oxidation in the carotenoid molecule) combined with the presence of a thick cyst wall. The stability of processed materials (disrupted cells that have been spray or freeze-dried) has also been shown to be significantly better than the synthetic product. This may prove to be a key commercial advantage for this product as the product (raw or in a feed) can be kept for longer without any significant decreases in its content. The 'potential bioavailability' of processed cells of Haematococcus has been determined using a simple in vitro assay (PI). Using this system, the efficacy of the processing procedures can be determined, prior to feed trials, P2 has examined the role of mechanical shear and enzyme/chemical digestion of the thick, sporopollenin-based, 9 algal cell wall. One process (patent application in progress), based on an enhancement of a mechanical fracturing process has been developed by P2. Characterisation of the products from this work was performed in conjunction with PI. PI examined a range of "off-the-shelf" mechanical digesters but these generally were found not to be suitable for astaxanthin-rich cells due to the high temperatures generated during the process. Similar results were also experienced with probe sonication and this was also rejected as an option. PI has employed a high pressure-fracturing/disruption system to break the cyst wall, rendering the contents (particularly astaxanthin) more bio-available for the fish or for further processing or extraction. No change to the carotenoid content or composition of cells during cell disruption or during subsequent spray-drying have been observed. This procedure can be scaled-up through pilot-scale to full industrial scale. SEM has been used to characterise the products and a full-scale pigmentation trial has been performed using two dried products (with P6). The stability of astaxanthin in freeze-dried zooid cells of Haematococcus has been performed by PI. In the presence of conventional industrial levels of ethoxyquin, losses over a 3 month period are <3%.
Conclusions: downstream processing of astaxanthin-rich cells of Haematococcus has been optimised and is suitable for scale-up development.
Task 3: Feed trials using astaxanthin-rich microalgae have been performed with both Rainbow Trout and Seabream (Gilthead and Black Spot) by P6 and P5, respectively. Detailed carotenoid analysis of muscle, skin and blood from pigmented fish produced by both sets of trials has been performed by PI. Individual pigment components and processed astaxanthin-rich cells of Haematococcus have been tested in comparison to a commercial synthetic product. Algal astaxanthin has been demonstrated to be an effective pigmentor of Rainbow Trout with no significant difference between the deposition rates or in the level of pigmentation attained of the algal product(s) and the synthetic formulation (P6 & PI). Both the algal and synthetic products attained marketable levels of pigmentation. The esterified forms of astaxanthin (mono- and di-esters) are utilised as well as the 'free' form. In addition, the astalanthin is deposited in the farmed fish as the optically active form (3S,3'S) which makes them indistinguishable from wild salmonids. The kinetics of plasma uptake and clearance, and the distribution of astaxanthin and its metabolites in the GI tract of Rainbow Trout have been determined by P6. The uptake of esterified astaxanthin is slower than that of 'free' astaxanthin indicating a delay due to hydrolysis of the esters. Studies on the deposition of pigments in the skin of Rainbow Trout have revealed that epimerization of (3S,3'S)-astaxanthin takes place (PI). This is not seen in the flesh.
Studies on the Gilthead Seabream and the Black Spot Seabream (both of which are pigmented in the skin and not the flesh), have shown that astaxanthin is not utilised very effectively in pigmentation of these fish. Indeed the studies indicate that lutein is the most effective pigment in Gilthead Seabream. These studies have revealed important information regarding the metabolism of carotenoids such as astaxanthin in seabream, information that could be used to formulate an improved pigmentor product. To compliment these in vivo studies on pigment deposition, P6 developed an in vitro assay to examine the factors regulating the uptake of astaxanthin (from alternative sources) using isolated, everted, fish guts. Fundamental data regarding the uptake of astaxanthin derived from the alga has been obtained.
Conclusions: The efficacy of algal astaxanthin (from Haematococcus) has been demonstrated for salmonid pigmentation against a synthetic commercial product.
Task 4: The AAPSTM system (designed by P7 and operated by PI) has been established as a means of mass algal cultivation. To date a number of algal species, including Haematococcus has been cultivated in this photobioreactor. The system retains it's air-lift operation but improvements in the design of the photostage and reservoir tank have resulted in improved performance (both physical and biological). The AAPSTM have been constructed at three working volumes: 65 L, 400L and 2000L. These photobioreactors can be operated indoors (optimised lighting systems have been developed and tested) and outdoors (using natural sunlight). The AAPSTM has been used to test alternative cultivation systems (batch, fed-batch and continuous operation). Both one-stage and two-stage . process for the cultivation of astaxanthin-rich cells developed at laboratory-scale has also been tested in the AAPSTM. Outdoor performance has been very effective, although more variable than indoor cultivation. P7 has applied for four GB patents to protect this invention. The Necton flat-plate flow through photobioreactor (P4) has been developed to improve overall performance and overcome some technical problems, such as cleaning and cooling evident when it was first operated. Cell yields in batch operation are close to that seen indoors in smaller cultures and astaxanthin is accumulated efficiently (25-99 mg/L). The flat-plate air-lift photobioreactor jointly developed by P3 and P9 has been operated by P9. It was designed so as to make best use of a short light path for effective light transmission to stimulate astaxanthin biosynthesis. Cell production was on a par with that seen in the laboratory for semi-continuous cultures of Haematococcus. Astaxanthin accumulation was however much slower than that reported by PI or P6 for outdoor cultures.
Conclusion: Production of astaxanthin-rich Haematococcus has been achieved in closed photobioreactors indoors and outdoors in three modes of operation, namely batch, semi-continuous and continuous. Optimal conditions and requirements for algal growth and pigment biosynthesis have been determined.
Task 5: A complete set of analytical methods and operating procedures for carotenoid analysis has been developed by PI. A methods manual was sent to all Partners in the first year of the project. Some additional methods have been introduced by P6 and PI. PI has instructed staff from other Partners in aspects of carotenoid analysis. PI has acted as a quality control check on the preparation of feeds by P6.
Conclusions: Specific methodologies developed and validated during the project have been utilised to support the main technical tasks (1-4).
Task 6: Overall project strategy based on technical issues and current state of the market was discussed at length at both the Annual meetings. A preliminary analysis of the current and predicted states of the market for astaxanthin and especially for natural sources of this carotenoid has been undertaken by P4 and P9. This was used to drive overall project direction and priorities. Two alternatives have been considered in detail - (a) production of algal astaxanthin as a pigmentor (primarily for aquaculture but also the poultry industry), and (b) production of a natural astaxanthin anti-oxidant product for use in human health food products etc. The former would be high volume, medium value whilst the latter would be low volume, high value. Using the technologies developed in the other tasks, opportunities to exploit both market sectors are envisaged.
Conclusions: The global market for astaxanthin is established and expanding. Two main areas for exploitation have been identified: fish pigmentation (especially salmonids) but also human nutrition health products.
Task 7: Project meetings have been held approximately every six months during the 36 months of the project. The first set of meetings focused on technical issues whilst the latter meetings also considered the strategic development of the project. Each meeting consisted of two sessions: first the technical presentation from each partner (a short oral presentation) followed by a business meeting. The Annual Meetings were particularly well attended. Minutes from these meetings were taken and distributed to all partners. A successful video-conference (two-site: UK and Portugal) was held.
Conclusions: A high level of participation was maintained. Inter-partner collaboration was very effective.
The vast majority of all partners have been able to fully utilise Email communication. Email, has been used to produce the Annual reports and distribute them to all Partners who are capable of using this medium. The Project web site has been established at http:cwis.livjm.ac.uk/carotenoid with the co-ordinator's institution acting as host for the site (PI). The site contains details of the project objectives and aims together with partner details and contact details for DGXII. An executive summary of the first two annual reports was placed on this site. An executive summary of the final report will be posted on the web site once accepted by DGXII. Overall, the level of communication between partners has improved greatly since the start of the project and, in general, attendance at the project meetings and inter- partner collaboration was very good.
Discussion
Overall, the vast majority of the project's original set of tasks (and sub-tasks) have been completed. Changes to the priority of certain sub-tasks have been made by the Partners at the various project meetings. For example, increased emphasis was placed on Task 2 (downstream processing) as it was clear that this was a critical step in the entire process. Less emphasis was placed on determining factors affecting fish health from the inclusion of dietary carotenoids. The project has determined the following
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