Detection of the oil-producing microalga Botryococcus braunii in natural freshwater environments by targeting the hydrocarbon biosynthesis gene SSL-3

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.

Large-scale screening of natural genetic resource in the hydrocarbon-producing microalga Botrycoccus braunii identified novel fast-growing strains

“…Frequent gene flow may also damp the power of natural selection. Hirano et al reported no genetic differentiation between tropical and temperate strains 31 , probably due to dispersal by birds and wind across a large geographic scale.…”

“…Fixed Effects Tests. a The Race (A, B, L, S) was estimated based on the molecular phylogeny of 18S rRNA sequences (See Hirano et al 31 ). b Number of parameters associated with the effect.…”

“…Since 2015 we have investigated a total of 112 natural ponds, including 50 ponds in tropical Indonesia and 9, 34, and 19 ponds in subtropical, warm-temperate, and cool-temperate Japan, respectively 31 , 32 . Surface water was collected using a phytoplankton net with 100 μm mesh to capture B. braunii , and a single colony was isolated by micropipette and incubated in a glass tube with a culture medium ( See Kawamura et al 32 for a detailed protocol).…”

“…A total of 180 wild strains were successfully isolated from 22 ponds located in various climate regions (Supplementary Table 1S ). The 18S ribosomal sequences were determined for 103 strains, and chemical races (A, B, L, S races) 34 were estimated based on the molecular phylogenetic tree 31 .…”

“…This study performed a large-scale screening of fast-growing strains from a total of 180 strains isolated from 22 ponds located in a wide geographic range, from the tropics to a cool-temperate climate. Although B. braunii is widely distributed in freshwater and brackish lakes, reservoirs, or ponds from temperate to tropical environments 15 , their natural densities are commonly quite low (10–10 2 colonies per L) 31 , which makes it difficult to find the alga in natural environments. We have developed a simple method for isolating B. braunii from the natural environments 32 and have newly isolated 70 wild strains from natural ponds in tropical Indonesia and temperate Japan 31 .…”