Abstract
The green microalga Botryococcus braunii produces hydrocarbon oils at 25–75% of its dry weight and is a promising source of biofuel feedstock. Few studies have examined this species’ ecology in natural habitats, and few wild genetic resources have been collected due to difficulties caused by its low abundance in nature. This study aimed to develop a real-time PCR assay for specific detection and quantification of this alga in natural environments and to quantify spatiotemporal variations of wild B. braunii populations in a tropical pond. We designed PCR primers toward the hydrocarbon biosynthesis gene SSL-3 and examined amplification specificity and PCR efficiency with 70 wild strains newly isolated from various environments. The results demonstrated that this PCR assay specifically amplified B. braunii DNA, especially that of B-race strains, and can be widely used to detect wild B. braunii strains in temperate and tropical habitats. Field-testing in a tropical pond suggested a diurnal change in the abundance of B. braunii in surface water and found B. braunii not only in surface water, but also at 1–1.5 m deep and in bottom sediments. This method can contribute to efficient genetic resource exploitations and may also help elucidate the unknown ecology of B. braunii.
Highlights
The green microalga Botryococcus braunii produces hydrocarbon oils at 25–75% of its dry weight and is a promising source of biofuel feedstock
We focused on a hydrocarbon biosynthetic gene, squalene synthase-like protein 3 (SSL-3)[21] to achieve the specific detection of B. braunii DNA
(4) we show the results of our real-time PCR-based quantification of spatiotemporal variations of a wild B. braunii population in a tropical pond and discuss how to use our method for future ecological studies
Summary
The green microalga Botryococcus braunii produces hydrocarbon oils at 25–75% of its dry weight and is a promising source of biofuel feedstock. This study aimed to develop a real-time PCR assay for specific detection and quantification of this alga in natural environments and to quantify spatiotemporal variations of wild B. braunii populations in a tropical pond. Fossils of B. braunii have been identified in organic remains of oil shales and petroleum source rocks[9], and their hydrocarbon oils are shown to be a major component of crude oils[10,11], indicating the noticeable contribution of this alga to petroleum generation Despite these remarkable characteristics of B. braunii, it has not yet been implemented in practical use for biofuel feedstock production. The objective of this study was to assess the applicability of a real-time PCR assay of the SSL-3 gene for specific detection and quantification of B. braunii (race B) in natural environments, by examining (1) the efficiency of DNA extraction, (2) the amplification specificity of PCR, and (3) the applicability to wild strains. (4) we show the results of our real-time PCR-based quantification of spatiotemporal variations of a wild B. braunii population in a tropical pond and discuss how to use our method for future ecological studies
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