Optimized medium conditions maximize colony regeneration from a single cell of Botryococcus braunii NIES836

Botryococcus braunii is a colonial alga recognized for its slow growth but high hydrocarbon accumulation. Although using genetic engineering to increase the growth rate and hydrocarbon yield of B. braunii is desirable, the presence of an extracellular matrix (ECM) significantly hinders the emergence of a homogeneous colony from a single DNA-transformed cell. Previously, we developed a method to isolate single cells without ECM from colonies. However, following the isolation of single cells, several months are required to regenerate colonies with a sufficient cell mass for subsequent analysis. To shorten the colony regeneration period, we investigated basal media and medium components, along with growth-promoting additives, in a series of single-factor experiments and optimized the concentrations of the medium constituents via response surface methodology (RSM). The results of the single-factor experiments revealed that the nitrogen source (a mixture of NaNO 3 and NH 4 NO 3 ), 1-naphthylacetic acid (NAA) and Fe(III)-citrate significantly increased the growth of B. braunii single cells into colonies. The optimal medium composition identified by RSM included 151.6 mg/L nitrogen source, 2.419 mg/L NAA and 15.3 mg/L Fe(III)-citrate. Verification experiments showed that the optimized medium resulted in a 1.75-fold increase in colony size compared with that of colonies grown in nonoptimized AF6 medium. This is the first report of the optimal medium composition for the regeneration of B. braunii colonies from single cells. • Colony structure of B. braunii prevents to obtain single-cell-derived transformants. • We succeeded to prepare and to regenerate B. braunii single cells. • New problem was how shortening the period to obtain cell mass enough for analysis. • We optimized the colony-regeneration medium using response surface methodology. • Colony size in the optimized medium achieved 1.75-fold larger than in AF6 medium.