Marine Biotechnology

The Marine Biotechnology Engineering Center (MarBEC), a NSF Engineering Research Center, was established in 1998 as a joint program with the University of Hawaii. MarBEC’s objectives were to provide the scientific and engineering underpinnings for the nascent marine biotechnology industry. It is anticipated that products from marine organisms will have a significant impact on the chemical, pharmaceutical, nutraceutical, cosmeceutical, food, feed and life-science industries. The target products of MarBEC included carotenoids, poly-unsaturated fatty acids (C-20, C-22 ω-3), UV-absorbing compounds and enzymes. The Center was highly interdisciplinary, at the interface of marine biology, chemistry and biochemical engineering, and supported a number of investigators and projects at both campuses. 


PRODUCTION OF CAROTENOIDS AND POLYUNSATURATED FATTY ACIDS

Carotenoids are isoprenoids containing a polyene chain of conjugated double bonds, which gives them a characteristic absorbance spectrum, and are derived from a basic symmetric acyclic C40H56 molecule. In plants and microalgae, xanthophylls are molecules of particular interest because they are involved in several photoprotective processes. In our studies, we have investigated the role of environmental factors, such as pH, temperature and light intensity, on the growth, pigment content and composition of the microalga Nannochloropsis gaditana, grown in a batch reactor under phototrophic conditions. 

The xanthophyll cycle consists of a cyclic reaction involving two successive de-epoxidations of violaxanthin to zeaxanthin via antheraxanthin, and the reverse epoxidation reaction. These two sequences are mediated by two different enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE), located on opposite sides of the thylakoid membrane. In Nannochloropsis only chlorophyll a is present; the main accessory pigments, violaxanthin and vaucheriaxanthin esters, play a major role in light harvesting. Optimum conditions for both growth and violaxanthin production were determined and treatment of cultures with iodoacetamide under high light conditions resulted in a 50% conversion of violaxanthin to zeaxanthin. We are also characterizing the growth and polyunsaturated fatty acid (PUFA) production in the marine microalgae, Glossomastix chrysoplastos, which belongs to a newfound class, Pinquiophyceae that produce a significant amount of lipids with high ω-3 PUFA content. 


HOST-GUEST CHEMISTRIES FOR PUFA SEPARATIONS

Recently it has been recognized that polyunsaturated fatty acids (PUFAs), especially ω−3 acids including eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), play an important role in human nutrition.  Both function not only as critical components of membrane lipids, but as unique precursors of certain hormones and regulatory metabolites (the eicosanoids). The primary source of EPA and DHA is marine microalgae and fish, in which PUFA concentrations can approach 40% of the total fatty-acid content. Extraction of lipids from the biomass is typically performed by solvent extraction, combined with either saponification or transesterification. A purification scheme with high selectivity is commercially desirable in light of their growing use in infant formulas. 

Production of pure component ω−3 acids is being examined using guanidinium disulfonate crystallization. This involves developing a hydrogen-bonded network for fatty-acid inclusion, composed of guanidinium and organodisulfonate ions. Hydrogen-bonded sheets, composed of sulfonate and guanidinium ions, are separated by the organic backbone of the disulfonic acid.  Inclusion cavities are formed between the sheets, with size and configuration controlled by the choice of the organodisulfonate pillar. Since this system was first reported (Ward and co-workers), modified structures have been synthesized, which incorporate organic molecules ranging from short-chain alkanes to large, multi-ringed aromatic compounds; no work to date has been reported in which complexes of long, linear molecules were synthesized. We are developing a guanididium-organodisulfonate network containing new organodisulfonate spacer geometries for the selective precipitation of linear, saturated fatty acids. We are currently synthesizing eight, ten, and twelve-carbon α, ω-alkanedisulfonates, and are investigating their inclusion behavior in the presence of six to twelve-carbon free acids and their methyl esters to obtain chain length specificity. 


CULTIVATION OF SPONGE CELLS 

Sponges have been found to be a diverse and prolific source of compounds of potential therapeutic value. However, the yield of bioactive compounds from sponges is typically low and the direct harvesting of sponges to generate sufficient quantities of bioactive materials is not feasible. The development of techniques for the in vitro production of these compounds would thus be a major advance. Our preliminary studies employ attachment-dependent mammalian cell culture techniques to develop sponge cell cultures. We have shown that the cells of the sponge Microciona prolifera are capable of attaching to the commercially available microcarriers, Cytoline 1 and Cytodex 3, and exhibit a degree of proliferation. Optimization of attachment conditions and media requirements of the sponges is ongoing.

Sponges associate with a rich microbial community. Libraries of small subunit ribosomal DNA (SSU rDNA)have been constructed for the identification of these microorganisms. Phylogenetic analyses were performed to determine phylogenetic type and origin. The 18S rDNA sequence of the sponge Axinella corrugata has been determined for use in cell line validation and for the design of molecular probes for the identification of sponge cells in culture. The consistent association of a crenarchaeote with the genus Axinella was verified. Similarities between the SSU rDNA sequences of clones obtained from genomic DNA extracted from the sponge revealed eubacteria that would not normally be found in oxygenic conditions. Further analysis is necessary to determine if a consistent association of these bacteria with the sponge exists and if they contribute to its metabolism and nutrition.

© Harvey Blanch 2013