Halogenated monoterpene production by microplantlets of the marine red alga Ochtodes secundiramea within an airlift photobioreactor under nutrient medium perfusion

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Abstract
Macrophytic marine red algae are a unique source of novel and bioactive terpenoids, including halogenated monoterpenes. Biomass and halogenated monoterpene production by regenerated microplantlet suspension cultures derived from the red alga Ochtodes secundiramea were studied within a perfusion airlift photobioreactor. Photobioreactor cultivations were carried out at 26°C, 140 μE m−2s−1 light intensity, 0.3 air L−1 culture min−1 aeration (3500 ppm CO2), and ESS/seawater medium perfusion rate of 0.2 L medium L−1 culture d−1. Macronutrient concentrations in the perfusion medium were adjusted to provide nitrate delivery rates of 0.0063, 0.077, and 0.74 mmol L−1 d−1 at a fixed N:P ratio of 19:1. Growth was maximized at the highest nutrient delivery rate, where 10 g dry biomass L−1 culture was achieved after 30 days of cultivation. GC‐MS analysis of dichloromethane extracts from cell biomass revealed that O. secundiramea microplantlets produced myrcene, three acyclic halogenated monoterpenes (10‐bromomyrcene, 10‐bromo‐7‐chloromyrcene, 3,10‐dibromomyrcene), and one cyclic halogenated monoterpene (6‐bromo‐1,2,8‐trichloro‐3,4‐ochtodene). 10E‐bromomyrcene levels were much higher than those of its isomer 10Z‐bromomyrcene, demonstrating stereoselective halogenation. Maximum yields of 10E‐bromomyrcene and 6‐bromo‐1,2,8‐trichloro‐3,4‐ochtodene were 15 and 13 μmol/g dry cell mass, respectively. Increasing the rate of nutrient delivery increased the accumulation of myrcene and 10‐bromomyrcene during the first 14 days in culture. Furthermore, the yield selectivity toward higher halogenated monoterpenes increased as the rate of nutrient delivery decreased. From this data, a biogenic scheme was proposed where cyclic and acyclic halogenated monoterpenes are derived from sequential halogenation of myrcene, their common precursor. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 415–428, 2003.