Geminating Chlamydomonas Zygospores for Lipid Extraction

Chlamydomonas is a common soil and water alga. Hundred of species are known but only a few are used routinely in laboratory research: C. reinhardtii C. eugametos and C. monoica. C. reinhardtii is the subject of more than 95% of the published Chlamydomonas-based research and is a well recognized model system for basic research in diverse areas of biology especially those using genetic approaches. Stress and in particular nitrogen starvation induces lipid accumulation a process that in C. reinhardtii requires an organic carbon source (acetate). C. monoica also accumulates intracellular lipid in response to nitrogen starvation but with no requirement for organic carbon. C. monoica does not and cannot use acetate as a carbon source to support growth in the absence of light. Instead the species is an obligate photoautotroph—an organism that requires light water and CO2 for synthesis of carbohydrates. These properties could be exploited commercially for lipid-related fuel and/or food production (including a variety of fatty acids and triglycerides). When environmental nitrogen reserves become limiting for growth C. monoica shifts from asexual growth to sexual reproduction. The vegetative cells differentiate into gametes that fuse to produce transient zygotes. Zygotes then mature into heavily walled dormant zygospores—awaiting the return of more suitable environmental conditions. The accumulation of lipids begins in the gametes and continues throughout zygote maturation. The cytoplasm of a mature zygospore is comprised primarily of numerous large lipid bodies that serve as an energy reserve. When environmental conditions improve including the return of adequate inorganic nitrogen the zygospores germinate releasing progeny cells that are capable of active vegetative growth as they consume the lipid reserves. Although the zygospore may provide the richest source of lipid from Chlamydomonas it is encased in a massive wall extraordinarily resistant to chemical or enzymatic degradation thus hampering experimental access to this oil reserve. This invention exploits the redistribution of lipid bodies to the zygospore progeny. At the time of germination progeny cells are released through slits in the surface layer of the spore wall which remains otherwise intact. One of the last stages in the germination process is the assembly of cell walls around the progeny. Because of the breaks in the residual zygospore wall and the naked nature of the progeny cells zygospores are extremely fragile just prior to progeny release. At this stage the progeny cells still retain most if not all of the lipid accumulated during spore formation. We have developed protocols to synchronize zygospore germination. This allows the inventors to harvest zygospores at this fragile stage. As anticipated the result was spore/cell lysis and release of lipid bodies into the surrounding culture medium. Applications: • Production of lipids or carotenoids for nutritional supplements • Production of lipids for alternative energy/biofuels • Production of lipids for organic or all-natural cosmetics