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reviews 6 Kwong, K. K. et al. (1992) l-'roc. Natl. Acad. &i. U. S. A. 89, 5675-5679 7 0 g a w a , S. et al. (1992) Proc. Nail Acad. Sd. U. S. A. 89, 5951-5955 8 Frahm, J. et al. (1994) N~1//R Biomed. 7, 45-53 9 Turner, R. et al. (1993) Magn. Reson. Med. 29, 277-279 10 Posse, S., Miiller-G~irtner, H-~X< and Dager, S. R. (1996) Sem. Clin. Neuropsychiatry 1, 76-88 11 H~mglSinen, M., Hari, R.., llmonimrfi, P,. J., Knuutila, J. and Lounasmaa, O. V. (1993) Rev. Mod. Phys. 65, 413-497 12 Lounasmaa, O. V. (1974) Experimental Principles and Methods Below IK, Academic Press 13 WiUiamson, S.J. and KaufmanrL, L. (1989) Brain Topog. 2, 129-139 14 HSm~il~inen, M., Hari, R., I]moniemi, P,. J., Knuutfla, J. and Lounasmaa, O. V. (1993) Rev. Mod. Phys. 65, 413-497 15 Damasio, A. P,. (1989) Neural Comput. 1, 123-132 16 Horwitz, B. (1994) Hum. Brair. Mapping 2, 112-122 17 Mclntosh, A. R.. and Gonzalez-Lima, F. (1994) Hum. Brain Mapping 2, 2-22 18 Drevets, W. C., Videen, T. O., Price, J. C., Preskorn, S. H., Carmichael, S. T. and P, aichle, M. E. (1992)J. Neurosci. 12, 3628-3642 19 Bench, C. J., Friston, K. J., Brown, P,.. G., Scott, L. C., Frackowiak, R. S.J. and Dolan, I(.J. (1992) Psychol.Med. 22, 607-615

20 Bench, C.J., Frackowiak, P,. s.J. aa~dDolan, R..J. (1995) Psychol. Med. 25, 247-261 21 Larisch, R., Klimke, A., Vosberg, H., L6flter, S., Gaebel, W. and Miiller-G~irtner, H-W. (1997) Neuroima,ge 5, 251-260 22 Berrios, G. E. and Bulbena-Villarasa, A. (1990) Psydzopharmacol. Ser. 9, 80-92 23 Aertsen, A. and Preissl, H. (1991) in Dynamics of Activity and Connectivity in Physiological Neuronal Networks in Non-linear Dynamics and Neuronal Networks (Schuster, H. G., ed.), pp. 281-302, VCH 24 Nelson, J. I., Salin, P. A., Munk, N. M.J., Arzi, M. and BuUier, J. (1992) Visual Neuro*ci. 9, 21-37 25 Taylor, J. G. The Racejbr Consciousness, MIT Press (in press) 26 Lohnson-Laird, P. N. (1983) Mental 3.¢odels - Towards a Cognitive Science of Language, Inference and Consciousness, Harvard University Press, Cambridge, MA, USA 27 Kant, I. (1787) Kritik der Reinen Vemunfi (Vol. 1) TheorieWerkausgabe Suhrkamp 28 Schmitt, F. O. (1978) in The Mindful Brain: Cortical Organization and the Group-Selective Theory of Higher Brain Function (Edelman, G. M. and Mountcastle, V. B., eds), pp. 1~5, MIT Press, Cambridge, MA, USA 29 Baxter, L. R.., Jr ez al. (1989) Arch. Gen. Psychiatry 46, 243-250

Cultivation of marine sponges for metabolite production: applications for biotechnology? Ronald Osinga, Johannes Tramper and Rene H. Wijffels The world's oceans harbour a large diversityof livingorganisms. As tropical rainforests have been searched for natural drugs, these marine organisms are being screened for useful products, and a number have been found in marine sponges. These are often produced only in trace amounts, and so a large quantity of sponges must be collected to obtain sufficient amounts of the target compounds. Hence, sustainable production of these compounds requires alternatives to harvesting sponge biomass directly from the sea, includingthe biotechnologicalproduction of sponge metabolites.

Sponges (taxonomically, members of the phylum Porifera) are the most primitive multicellular organisms within the animal kingdom. Their body structure is simple (Figs 1 and 2) and differs from that of other multicellular animals by ~:he absence of organs and true tissues. Sponges corttain specialized ceils with different shapes and funct:ons, but these ceils are not R. Osinga ([email protected]), J. Tramper and R. H. Wijffels are at the Wagenin~en Agricultural University, Department of Food Science and Technoi'ogy, Food and Bioprocess Engineering Group, P O Box 8129, 6700 EV, Wageningen, The Netherlands. TIBTECH MARCH 1998 ('COL 16)

differentiated. Each cell can, in principle, reversibly alter its function. The most characteristic feature of the sponge body is the so-called aquiferous system, which is a network of channels and chambers through which water flows continuously (Fig. 1). This water current is generated by flagellated cells (choanocytes) that line the walls of the chambers. The space between the channels and chambers is filled with a gelatinous matrix containing free-floating cells and two kinds of skeletal material (spicula and spongin, Fig. 1); this part of the sponge body is called the mesohyl.

Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0167 - 7799/98/$19.00. Pll: S0167-7799(97)01164-5

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reviews Sponges are sessile organisms, which means that they are attached to a solid substratum (rock, coral, etc.). Their food consists of small organic particles (microalgae, bacteria and dead organic matter). Most of these particles are captured from the inflowing wa:er by a collar of microvilli that surround the flagella of the choanocytes. Captured particles are transported to cells in the mesohyl, which digest the material and produce metabolic energy. Metabolic waste products, such as ammonia and organic compounds, are released in the aquiferous system and removed from the sponge body with the outflowing water. More detailed information on general sponge biology has been reviewed by Bergquist I and Simpson2.

Sponge products A wide variety of interesting new compounds and classes of compounds have been isolated from sponges 3-5. The structural diversity of sponge secondary metabolites is larger than that oJ7 any other marine phylum. Furthermore, sponges have a high strike rate, especially for cytotoxic compounds: more than 10% of the sponge species investigated show cytotoxic activity, which is considerably higher than other marine animals (2%), terrestrial plants (< 1%) or microorganisms (< 1%)4. Some of these cytotoxic compounds may have potential as anticancer drugs. In addition to cytotoxins, other compounds with antibiotic, antiviral, anti-inflammatory and cardiovascular properties have been found in sponges, as have compound,; that can be used as antifouling agents in marine technalogy. To date, the limited availability of sponge products has hampered their progress towards cormnercial production. For example, Halichondrin B, a complex molecule first isolated from the sponge Halichondria okadai, has currently reached the stage of clinical evaluation but has not yet advanced to clinical trials, owing to the limited supply of sponge material'L Chemical synthesis or biotechnological production of sponge metabolites may provide a solution to this problem. Three important questions that have to be addressed when designing a production method fi)r a sponge metabolite are how, why and where the ~ponge produces the compound of interest4. Knowing the biosynthetic pathway of a natural compound can help to develop a chemical-synthesis method; for biotechnological production of a compound, knowledge of the ecological factors influencing this production will be essential. For instance, cytotoxic compounds are probably chemical weapons used in the corrLpetition for space with other sessile life forms, and antibiotics will be produced to prevent the growth of microorganisms on the sponge's surface or of microorganisms that enter the body via the aquiferous system. Cocultivation of the target organisms may be needed to maintain the production of these chemical weapons in a bioreactor. The interesting compounds may also be produced by endosymbiotic bacteria that live in the sponge mesohyl. A well-studied example of this is the production of antibiotic polybrominated biphenyl ethers by the endosymbiotic cyanobacterium Oscillatoria spongeliae 7-9,

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~

P

Figure 1 Body structure of a simple asconoid (vase-shaped) sponge. (a) Habitus (external appearance). (b) Cross-section, showing the aquiferous system (arrows indicate the direction of the water current). (c) Detail of a channel, a flagellated chamber and the mesohyl. Abbreviations: p, pinacocyte (skin cell); c, choanocyte (flagellate cell generating the water current through the sponge body); a, archaeocyte (free floating cell); s, spongine (collagenous fibres); sp, spicula (needle-like structures, made of either silica or calcite). All choanocytes have a collar of microvilli.

which is hosted by the Indopacific sponge Dysidea herbacea. The biphenyl ethers can make up to 12% of the sponge's dry weight 8, which makes the sponge mesohyl a very unsuitable medium for the growth of bacteria other than O. spongeliae. Finally, a sponge may produce and excrete the target compound continuously, or it may only contain a constant amount of compound per unit volume. In the latter case, the sponge biomass must be harvested to obtain the compound of interest, while in the former case, living sponge biomass can be used as a production plant.

Production o f sponge biomass Commercial biotechnological production of sponge biomass started in the late 19th century, when the first techniques for in situ aquaculture of natural bath sponges (Spongillidae) were developed 1°. The basis for these early techniques was the undifferentiated TIBTECH MARCH1998 (VOL 16)

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reviews published an extensive review on the early spongeaquaculture techniques I1, and these descriptions are still the basis for present-day in situ bath-sponge production (Fig. 3). In N e w Zealand, progress is now also being made with aquacultures of sponges for metabolite production 12. Aquacultures have the disadvantage of being dependent on the in situ conditions, which may often be suboptimal for growth. Barthel and Theede have successfully grown cuttings of the sponge Halichondria panicea in semienclosed, temperature-controlled systems that were continuously supplied with unfiltered seawater ~3. Additional nutrition (cultured Chlorella) was regularly added to the cultures, which increased the growth rates of the sponges considerably. In a similar system, the freshwater sponges Ephydatia fluviaitilis and Spongilla alba were fed with different concentrations of Escherichia coil The growth rate of the sponges showed a positive correlation with bacterial concentrations in the f o o d 14. These semicontrolled cultivation methods definitely have great biotechnological potential, but are still pardally dependent on unpredictable external factors. Therefore, the possibility, of producing sponge biomass under completely controlled conditions is worth investigating, tKelevant aspects for bioprocess engineering, such as the nutritional demands of the sponges, their growth rates and the yields of sponge biomass and secondary metabolites, should be emphasized in further studies. Figure 2 The Indopacific sponge species Theonella conica (photograph taken by R. Roozendaal and provided by R. W. M. van Soest).

Figure 3 An in situ culture of Spongia oHicinalis in the northwestern Mediterranean Sea. Cuttings of the sponges are connected to ver':ical ropes (photograph provided by J. Vacelet).

character of the sponge cells, which gives sponges a strong regenerative power. Small parts of a sponge can regenerate to full-grown adult individuals. Hence, cutting an adult sponge into pieces will produce many genetically identical new sponges. In 1910, Moore TIBTECHMARCH1998 {VOL16)

Sponges in closed systems Sponges are difficult to keep in vitro 15,16. In their natural habitat, sponges are supplied with an almostunlimited amount of unfiltered seawater that provides food particles and removes waste products. In a small enclosure, food sources will rapidly be depleted and waste products will accumulate. Accumulation of waste products and toxins is a c o m m o n problem when aquatic animals are cultivated in dosed systems. The accumulation of nitrogenous wastes (e.g. ammonia and nitrite) can be especially harmful to the animals 17. A more specific problem with sponges in enclosed systems is the supply of an adequate food source. Because sponges are unselective filter feeders, their food consists of a mixture of living microorganisms, dead organic particles and dissolved organic matter. Therefore, it may be difl3cult to create an artificial mixture of food particles that can cope with their nutritional demands Is. To the best of our knowledge, the only successful long-term culture of a marine sponge in a closed system was a culture of Ophlitaspongia seriatais. These sponges were fed with a mixture of four microalgal species of different sizes, combined with a dead bacterial culture (containing both intact bacterial cells and small particles from disrupted cells). To date, it is unknown whether sponges can be grown on a food source consisting of a single species. In our laboratow, we have managed to maintain two in vitro sponge cultures for more than one year. The tropical demosponge species Pseudosuberites andrewsi was cultured on a diet of Chlorella vulgaris and Dunaliella sp. and the

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Sparger Figure 4 Possible design of a sponge bioreactor. The sponges are attached to a solid substratum in a well-mixed air-lift reactor. A continuous culture of microalgae provides a continuous supply of food for the sponges. The bioreactor water is recirculated through the algal culture in order to prevent the build up of inorganic nutrients in the bioreactor: these inorganic nutrients are assimilated by the algae.

temperate boring sponge Cliona celata was cultured with ChlorelIa vulgaris as the only food source. However, no significant increase in sponge biomass was observed in either culture (R. Osi;aga et al., unpublished). More than 95% of sponge species use silicon to produce skeletal material (spicules). Siliceous spicules can contribute significantly to the total sponge biomass. For instance, spicular silica contents of up to 62.3% of the dry weight have been found in species of the order Haplosclerida, and up to 74.9% in species of the order Petrosiida 19. Hence, the availability of silicon may easily become growth limiting in a small enclosure. As it can be taken up by the sponge cells as dissolved silicon, silicon limitation may be simply prevented b7 adding a silicon-containing salt to the medium in the bioreactor. The size of the addition should be deduced from the sponges' silicon-uptake rate, which can easily be determined 20.

Sponge-cell cultures Cultures o f animal cells are being widely used to produce pharmaceuticals on an industrial scale 21. Some attempts have been made to produce continuous cell lines of sponges for the biotechnological production of sponge metabolites 22~23 but, so far, only primary sponge-cell cultures have been established. ]'he primary cultures were maintained in a medium consisting of different commercial media, calcium- and magnesiumfree artificial seawater, phosphate buffer 22 and foetalcalf serum. Calcium- and magnesium-free seawater was used because these metal ions play a role in the reaggregation process of sponge cells, which is undesirable in cell cultures. Antibiotics had to be added continuously to the cultures, not only to obtaba axenic

sponge cells, but also to kill the endosymbiotic bacteria that are released when a sponge cell dies. There are still some important problems to overcome in the progress towards the continuous culture of sponge-cell lines. The rich media imply a high risk of microbial contamination 2e,23 and the sponge cells can easily be confused with unicellnlar eukaryotes 24. Moreover, cell proliferation did not occur without the addition of mitogenic agents 22,23. In addition to these practical problems, the following questions should be addressed before starting the process of producing a sponge-cell culture: will a sponge cell still produce the target compound under completely controlled conditions, in the absence o f enviromnental f~ctors and without their endosymbionts? Conversely, will sponge-endosymbiont cultures still produce the target compounds in the absence of the sponge host?

Designing a sponge bioreactor The cultivation of intact sponges or sponge cuttings (aggregates) in bioreactors seems to have some advantages over cell cultures. First, simple and relatively cheap media can be used (seawater with algae and/or bacteria), and the cultures do not have to be kept sterile; the addition of some antibiotics may, however, be necessary to prevent the growth of parasitic fungi and bacteria t4. Second, these systems will more closely resemble natural conditions. Sponge-endosymbiont relations are more likely to remain intact, decreasing the probability of the sponges losing their ability to produce the metabolites of interest. H o w should a bioreactor for sponge (aggregate) cultivation be designed? In addition to the important factors mentioned above (an adequate food source, addition of silicon and removal of ammonia and other TIBTECHMARCH1998 (VOL 16)

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reviews toxic compounds), some other engineering aspects need to be considered. One o f these is mixing, which will augment mass transfer in the bioreactor and will keep the food particles in suspension. Deposited particles will not only be lost for consumption by the sponges, but they will also be degraded by bacteria, consuming oxygen and producing waste products that decrease the water quality. The oxygen supply to the medium in the bioreactor is also important. Many sponges are sensitive to hypoxic conditions, which reduce their growth rate (D. Barthel, pers. commun.). Hence, in addition to strong mixing, effective aeration of the bioreactor should also be established. Accumulation of ammonia and other metabolic wastes in the bioreactor can be prevented either by a rapid, continuous renewal of the water or by using a recirculation-biofiltration system. A biofilter is usually a packed bed in which immobilized bacteria degrade the hazardous compounds. Ammonia (the most important metabolic waste product), for instance, is converted to nitrate by nitrifying bacteria. In order to construct a suitable biofiltration system for a sponge culture, the anunonia production of the sponges, as well as the capacity of the filter to remove it, should be determined. l~ecirculation systems have the advantages that they consume less water than flow-through systems and that food particles can be maintained inside the system. A prerequisite for this is that food particles must be able to pass through the fdter. A possible alternative to the packed-bed biofilter could be a recirculation system in which the bioreactor water is recirculated through a culture o f microalgae. In this case, the algae will act as both a food source for the sponges and a biofilter, by assimilating the inorganic nitrogen compounds (both ammonia and nitrate) (Fig. 4). One disadvantage of recirculation is that lessbiodegradable toxins and ~rganisms that cause sponge diseases (viruses, bacteria or fungi) will remain inside the system.

Future prospects So far, the screening of the marine biosphere for interesting compounds has given promising results 25. However, the pathway from discovery to commercial production is long and difficult. In the case of sponge metabolites, the lack of sufficient sponge material has hampered progress towar(ts the biotechnological production of these compounds 6,22. Although the need for methods to cultivate sponges or sponge cells has been recognized, little work ha,; been done to establish such cultures. Research on sponge-cell cultures is still at a preliminary stage and the media required for spongecell culture imply high costs. However, if continuous cell lines can be established, some valuable products may be produced in this way. At present, most progress has been made with in situ cultivation of sponges. This is an economically feasible option for the product:ton of sponge metabolites because it is a cheap and relatively easy method to TIBTECHMARCH1998 (VOL 16)

obtain sponge biomass. The promising results with semienclosed systems suggest that the cultivation of sponges or sponge cuttings under completely controlled conditions will also be possible in the near future. Research should focus on the factors that limit or inhibit the growth of sponges in closed systems. Determining these factors will open the way to the commercial production of the numerous important sponge metabolites.

Acknowledgments We thank R.. W. M. van Soest and J. Vacelet, who kindly provided us with pictures of sponges and sponge maricultures.

References 1 Bergquist, P. R. (1978) Sponges, Hutchinson 2 Simpson, T. L (I9841 The Cell BiNogy qfSponges, Springer 3 Ireland, C. M., Copp, B. i%., Foster, M. P., McDonald, L. A., P, adisky, D. C. and Swersey, C. (1992) in Marine Biotechnology, Pharmaceutical and Bioactive Natural Products (Attaway, D. H. and Zaborsky, O. R.., eds), pp. 1-43, Plenum 4 Garson, M.J. (19941 m Spongesin Time and Space(Van Soest, i%. W. M., Van Kempen, T. M. G. and Braekman, J. C., eds), pp. 427-440, Balkema 5 Schmitz, F.J. (1994) in Sponges in Time and Spar (Van Soest, R. W. M., Van Kempen, T. M. G. and Braekman, J. C., eds), pp. 485-496, Balkema 6 Munro, M. H. G., Blunt, J. ~,V, Lake, P,. J., Litaudon, M., Battersb.ill, C. N. and Page, M.J. (1994) in Sponges in Time and Space (Van Soest, 1~. W. M., Van Kempen, T. M. G. and Braekanan,J. C., eds), pp. 473-484, Balkema 7 Unson, M. D. and Faulkner, D.J. (1993) Experientia 49, 349-353 8 Unson, M. D., Holland, N. D. and Faulkaler, D.J. (1994) Mar. Biol. 119, 1-11 9 Hinde, 1<., Pironet, F. and Borowitzka, M. A. (1994) Mar. Biol. 119, 99-104 10 Snfith, H. M. (1897) Fish Commision Bulletin 15,225-240 11 Moore, H. F. (1910) Bull. US Bur. Fish. 28, 545-585 12 Battershill, C. N. and Page, M. J. (1996) Aquacuh. Update Spring, 56 13 Barthel, D. and Theede, H. (19861 Ophelia 25, 75-82 14 Poirrier, M. A., Francis, J. C. and LaBiche, R. A. (19811 Hydrobiologia 79, 255-259 15 Kinne, O. (1977) in Marine Ecology (Vol. 3, Pt 2) (Kimle, O., ed.), pp. 627-641, Wiley 16 Foss~t, S. A. and Nilsen, A.J. (19961 KorallenrjffAquarium (Vol. 5), pp. 35-65, 8chmettkamp 17 Colt, J. E. and Armstrong, D. A. (19811 in Proceedings qfthe Bioengineering Symposium fiv Fish Culture (Allen, L.J. and Kinney, E. C.. eds), pp. 34-47, American Fisheries Society 18 Fry, W. G. (1971) in Proceedingsof the Fourth European Marine Biology Symposium (Crisp, D. J., ed.), pp. 155-178, Cambridge UniversiD Press 19 Desqueyrouz-Faundez, R.. (1990) in New Perspectives in Sponge Biology (K. R.iitzler, ed.), pp. 279-283, srmthsonian Institution Press 20 Frohlich, H. and Barthel, D. (1997) Mar. Biol. 128, 115 125 21 Klausner, A. (1993) Biotechnol. 11, 35-37 22 Pomponi, S. A. and Willoughby, R.. (19941 in Sponges in Time and Space (Van Soest, I<. W. M . Van Kempen, T. M. G. and Braekman, J. C., eds), pp. 395-400, Balkema 23 [lan, M., Contini, H., Carmeli, S. and R.inkevich, B. (1996)J. Mar. Biotechnol. 4, 145-149 24 Custodio, M. P,., hnsiecke, G., Klautau, M., Pdnkevich, B., P,.ogerson, A. and Mtiller, W. E. G. (1995)J. Eukaryot. Microbiol. 42, 721-724 25 Cowan, D. A. (19971 Trends Biotechnol. 15, 129-131