AIR2-CT94-1283 Biomass Production System For The Biosynthesis Of Carotenoids

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AIR Cluster III - Bioconversion : Biological Conversion : Fine Chemicals : Pharmaceuticals/Cosmetics : Process Engineering : Vegetable Oil/Fat

	Proposal No:	AIR2-CT94-1283
	Date Prepared:	October 1996, April 1998
	Source:	First Project Progress Report Progress Reports and Final Report Summary

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First Project Progress Report

INTRODUCTION

This project is investigating the feasibility of large scale carotenoid production using microalgae such as Dunaliella and Haematococcus species. Studies cover both closed photobioreactors and raceway ponds, under the conditions found in Southern Europe. The research being carried out covers a number of interdependent tasks including laboratory scale cultures, process scale up and optimisation, as well as analysis and process control, system support and management. The anticipated final result is a complete cost effective production system for the biosynthesis of natural carotenoids integrating all the laboratory to pilot plant steps, as well as the knowledge to operate the system for the production of different carotenoids using strains of different microalgae selected. The final product may be produced either as a microalgae powder or as extracted carotenoids in vegetable oil solution.

OBJECTIVE

This project aims to improve the biosynthesis of carotenoids through the development of a new environmentally friendly technology, cost effective production of natural carotenoids, which are increasingly important pigments and anti-oxidants widely used in Agro-food, pharmaceutics, cosmetics and Mealth-Food Industries. Different technologies for the production of microalgae biomass will be combined integrating two different systems:

1. the photobioreactors, where there is a fairly good knowledge in Europe (Britain, France and Italy), with pre-competitive results already described, which will be considered, and

2. the raceway pond systems, where extensive experience was developed in Israel and U.S.A., with commercial production facilities in operation, with whom technology transfer contacts have been initiated. The whole system, pioneered in Europe, will be applied to beta-carotene production using Dunaliella, and the photo-bioreactor, will be used for astaxanthin biosynthesis with Haematococcus.

The research will consist of a set of Tasks grouped into four Macro-Tasks:

1. MT l: Laboratory Scale Cultures;

2. MT 2: Process Scale up and Optimization.

3. MT 3: Analytical Development and Control.

4. MT 4: System Support and Management.

Those Macro-Tasks are interdependent and will be executed in parallel using a PERT approach for the actual management and control. All participants have a significant contribution for each of the Macro-Tasks which aims to fulfil a specific milestone. The expected results are:

1. a full cost-effective production system for the biosynthesis of natural carotenoids integrating all the laboratory to pilot-plant steps;

2. the knowledge to operate the system for the production of different carotenoids using different microalgae selected strains; and

3. the obtaining of high quality natural products, either in the form of microalgae powder or extracted carotenoids in vegetable oil solution.

RESULTS

During the first year of work, the four laboratories participating in the project assembled laboratory scale culture equipment and built small scale raceway ponds. Medium scale ponds are already under use in Lisbon and the Algarve.

The analytical techniques to be used during the project have also mostly been set up, problems being found in terms of the very salty media used with Dunaliella. Development of protocols for carotenoid analysis has also caused problems.

One of the important points under consideration is the selection of the best strains in terms of growth and carotenoid production under the climatic conditions found in the south of Portugal. Some progress in this area has been made, but this remains a major concern. The development of culture conditions is also important, in terms of favouring growth of the chosen strains and, at the same time, discouraging growth of contaminants. It has already been concluded that carotenoid production has to be carried out under a different set of conditions from those favouring increase in biomass. Therefore the optimisation of both sets of conditions is necessary.

The separation of the cells, has also been a subject under study, with some innovative methods being tested. These include microfiltration and the natural migration of cells from a layer of high salt concentration to a less concentrated one.

The extraction of the carotenoid from the cells is also complex; experiments using supercritical extraction as well as conjugation of flocculation/oil extraction have been started. The best choice was found to be the extraction into vegetable oil. The important problem of preserving the carotenoids either within or outside the cells has also been investigated, with some promising results.

CONCLUSIONS

Although, during the first year of the project, important steps have been taken towards the economic production of carotenoids from microalgae, these are still mostly of infrastructural nature. This includes the installation of laboratory growth equipment and small and medium size raceway ponds. Various analytical procedures and technological operations have also been developed and will be used during the continuation of the project.

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Progress Reports and Final Report Summary

Introduction
This project has as its main objective the investigation of the microalgal production systems for carotenoids, using both Dunaliella and Haematococcus strains, in both photobioreactors and raceway ponds. The project consists of four main tasks, each of which is further subdivided. The four main parts are :

• establishment of laboratory scale cultures.
• scale-up.
• development of analytical determination methodologies and their use in process control
• system support and project management and control.

Another objective of this project is the identification of environmental and other conditions necessary to permit the development of microalgal cultivation for the production of carotenoids in Europe. In achieving this objective, the main activities aim to:

• identify and evaluate conditions necessary for astaxanthin biosynthesis by the alga Haematococcus spp.
• develop protocols and provide support concerning the analysis of beta-carotene, astaxanthin and other relevant carotenoids accumulated by the microalgal species under investigation, with special reference to the analysis of geometrical isomers of carotenoids.

The project was mainly focused upon the methodologies which are necessary to mass produce useful carotenoids from microalgae, under the climatic conditions found in Europe. The carotenoids which were sought were astaxanthin and beta-carotene. These find use in fish-farming, cosmetics and pharmaceuticals. The objective here was to use biotechnological means as an alternative to existing organic chemical synthesis routes. These biological routes may find favour, popularity and appeal, due in part to the claim made as natural products. These methods also give a high content of cis isomers which are not obtained chemically.

Activities
This work focused on three main areas of activity:

• determination of environmental controls on the biosynthesis of cis- and trans-isomers of beta- and alpha-carotene in Dunaliella.
• downstream-processing of Dunaliella and Haematococcus
• continuous cultivation and astaxanthin synthesis by Haematococcus in a Chemostat.

The main achievements from year 3 of the project are as follows:

• a thorough understanding of the environmental effects of carotenoid biosynthesis in Dunaliella, Haematococcus and Dysmorphococcus;
• the optimisation of downstream processing conditions for algal beta-carotene and astaxanthin (harvesting, spray- and freeze-drying, storage conditions)
• requirements for successful scale-up of algal growth systems (small ponds for Dunaliella; air-lift and chemostat for Haematococcus) have been determined for batch, semi-batch, draw-fill and continuous cultures.
• photoautotrophic, mixatrophic and heterotrophic growth potential and carotenoid production has been determined.
• a complete set of analytical protocols for the analysis of carotenoids and their isomers has been developed .

Methods
The various partners in the project used different types of photobioreactors, which varied in volume from a few millilitres (to study a large number of variables in a short time), to several cubic meters (used for biomass production). At the larger scale, glass air-lifts (for Haematococcus) and either polyethylene sleeves or open-air ponds (for Dunaliella) were used. The latter were of the raceway type with a culture depth of 20 cm. These could be protected by greenhouse systems, that were important in terms of temperature maintenance. The small and medium ponds were fitted with data acquisition systems, which allowed the retrieval or storage of data from different sensors. The air delivered to the system was enriched in carbon dioxide, which was important in terms of the photosynthetic process and also for the pH control.

The harvesting of the cells from the culture media is one of the crucial steps for the economical viability of the process. Different methods were tested including flocculation, centrifugation, flotation and tangential cross-flow filtration. Flocculation gave positive results, although such methods may be unsuitable, as the flocculating chemicals become an impurity to the product, which is supposed to be natural. Centrifugation is an efficient method but involves high costs, both in investment and in running costs, due to the high volumes that must be dealt with. Flotation did not prove to be adequate due to the high investment costs and energy requirements. Tangential cross flow filtration may be a potentially interesting method. This was successfully tested at laboratory scale but still needs appropriate development for large-scale application. For the high saline medium of Dunaliella salina, a pre-concentration step which involved the migration of the cells to an added less saline layer, facilitated the following harvest.

For extraction of beta-carotene from the harvested cells of Dunaliella salina, three different methods were used. One was impact homogenisation at room temperature, producing a beta-carotene rich oil. The second process involved increasing the temperature above 70 degrees centigrade; where beta-carotene was again obtained in the extracting oil, although some degradation occurred. Finally, supercritical carbon dioxide extraction was also tested. It was noted that these extracts showed a very significant increase of the cis/trans ratio, when compared with that of the mixture initially put into the extractor. This may be of economical interest as the cis isomer is more easily absorbed by the human organism than the trans. However, supercritical extraction may still need further development for a large-scale application.

Micro-encapsulation of spray-dried Dunaliella and Haematococcus was undertaken. It was found that the most appropriate material for this purpose was maltodextrin.

All algal materials were tested for carotenoid stability. Beta-carotene and astaxanthin recoveries were high even at the higher temperatures used. Recoveries of the carotenoids by freeze-drying were generally better than those obtained by spray-drying. Stability of the materials obtained was high with only a 10 % loss over a 90 days period at room temperature in the dark and in the presence of air. Sun-dried or heat-dried samples of Dunaliella were rapidly degraded in terms of beta-carotene concentration. On the other hand, flocculated and centrifuged moist Dunaliella kept in refrigerator in the absence of oxygen showed only a 15 % degradation over nine months.

FDA standard methods and ELISA tests were used for the detection and identification of possible contamination with bacteria or toxins. In fact, no toxins or fungi were ever detected. Protozoa present in Dunaliella cultures are still to be fully identified.

The recycling of the saline medium used for the growth of Dunaliella was studied, as its composition involves the consumption of high quantities of salt. It was found that by recycling the growth medium three times, productivities of beta-carotene in the effluent from centrifugation and from flocculation were similar and sometimes higher than those obtained in the control medium. When the organic content of the medium reached high values (COD's higher than 800 mg/l), it was necessary treat it. By supplementing that effluent with a nitrogen source, it was possible to reduce the organic concentration by 65 %, using extreme halophytic bacteria. The resulting effluent will still need further treatment in a maturation pond before being discarded to the sea. A further reuse of this treated effluent has still to be studied.

Results
Scale-up of Dunaliella production/harvesting/processing and media recycling from laboratory-scale to production-scale ponds has been achieved with Dunaliella salina resulting in biomass production of human consumption grade, with good stabilisation of the beta-carotene. The harvesting of biomass is an important step in the complete production process. Comparison between centrifugation, flocculation and was found to be a very attractive method where pre-concentration resulted in a significant volume reduction of biomass. The biomass was free from contaminant chemicals. Scale-up of Dunaliella production/harvesting/processing from pilot-scale to production-scale ponds was linked to evaluation of end-product use and assessment of market requirements. These studies related to harvesting and carotenoid isolation, stabilisation and packaging and enabled optimisation of Dunaliella growth and beta-carotene production in production pounds.

Work has centred upon the strategies for mass production of Dunaliella salina and Haematococcus pluvialis, (the main sources of beta-carotene and astaxanthin respectively), and on the monitoring and evaluation of productivity in order to optimise production systems. The work resulted in the selection, isolation and improvement of strains of Dunaliella salina and Haematococcus pluvialis. These were either obtained from culture collections or isolated locally. The optimised cultures were those which had rapid growth, good carotenoid production and were well adapted to the local climatic conditions in Southern Europe where higher light intensities and sunshine hours favour the mass production of microalgae.

The objective of mass carotenoid production involved the optimisation of growth culture media and of the conditions for carotenogenesis. It was found that different conditions have to be used for cell growth and carotenogenesis. For the latter, environmental stresses were needed, such as exposure to high (or low) photon flux densities, extremes of temperature or nutrient deprivation.

The characteristics of Dunaliella salina production in an open air ponds plant are reasonably friendly in terms of the environment. In fact, the production ponds are to be built in an area of old salt marshes - which allows the local production of the large quantities of salt required. Therefore the type of construction and use of the ponds is entirely integrated in the natural conditions of the area. Otherwise there are no objections in terms of air or sound pollution and no foreseeable negative impact on the local native animals.

Discussion
The carotenoids are of fundamental importance to life. Not all their functions are fully understood, but it is evident that carotenoids are intimately involved in the protection of organic molecules from oxidative destruction and in light-induced energy production via photosynthesis. The beta-carotene can be extracted from natural sources (like carrots or microalgae) or produced from petrochemicals. From an analytical point of view, the chemical synthetic beta-carotene differs from the natural form in its isomers composition. The synthetic molecule has the all-trans form while the natural molecule is a mixture of cis (mainly 9-cis and 15-cis) and trans isomers. While carrots have almost only the trans isomer, Dunaliella salina may reach 60 % of cis, depending on the culture conditions. There is evidence that the natural , beta-carotene, for its isomeric composition, is better adsorbed by living organism s. Hence, the biological beta-carotene is normally targeted to dietetic, pharmaceutics and cosmetic markets. The world market for beta-carotene is evaluated at dollars US 150 to 250 millions per year, corresponding to a production of 300-400 tons of which the natural products account for about 10 %. The size of this market does not seem to be growing at this moment.