Hydrocarbon productivities in different Botryococcus strains: comparative methods in product quantification

Abstract

Six different strains of the green microalgae Botryococcus belonging to the A-race or B-race, accumulating alkadiene or botryococcene hydrocarbons, respectively, were compared for biomass and hydrocarbon productivities. Biomass productivity was assessed gravimetrically upon strain growth in the laboratory under defined conditions. Hydrocarbon productivities were measured by three different and independent experimental approaches, including density equilibrium of the intact cells and micro-colonies, spectrophotometric analysis of hydrocarbon extracts, and gravimetric quantitation of eluted hydrocarbons. All three hydrocarbon-quantitation methods yielded similar results for each of the strains examined. The B-race microalgae Botryococcus braunii var. Showa and Kawaguchi-1 constitutively accumulated botryococcene hydrocarbons equivalent to 30% and 20%, respectively, of their overall biomass. The A-race microalgae Botryococcus braunii, varieties Yamanaka, UTEX 2441 and UTEX LB572 constitutively accumulated alkadiene hydrocarbons ranging from 14% to 13% and 10% of their overall biomass, respectively. Botryococcus sudeticus (UTEX 2629), a morphologically different green microalga, had the lowest hydrocarbon accumulation, equal to about 3% of its overall biomass. Results validate the density equilibrium and spectrophotometric analysis methods in the quantitation of botryococcene-type hydrocarbons. These analytical advances will serve in the screening and selection of B. braunii and of other microalgae in efforts to identify those having a high hydrocarbon content for use in commercial applications.

Fig. 1

Botryococcus cells, grown in 500 mL of modified Chu-13 medium in conical Fernbach flasks upon orbital shaking. Note that (a) Botryococcus braunii var. Showa, (b) Botryococcus braunii Kawaguchi-1, (c) Botryococcus braunii Yamanaka, (d) Botryococcus braunii UTEX 2441, (e) Botryococcus braunii UTEX LB572 micro-colonies centrifuging to the center of the H₂O-based growth medium, whereas (f) Botryococcus sudeticus (UTEX 2629) cultures made uniform suspension

Fig. 2

Cumulative biomass productivities of Botryococcus strains in continuous fed cultures. Data points indicate the time when a fixed fraction of the culture (40% of the culture volume) was harvested and replaced by an equal volume of fresh growth medium. Cells were grown in 500 mL of modified Chu-13 medium in conical Fernbach flasks upon orbital shaking. The slopes of the straight lines defined the corresponding rates of biomass accumulation, equal to (a) 125 mg dw L⁻¹ d⁻¹ for Botryococcus braunii var. Showa, (b) 80 mg dw L⁻¹ d⁻¹ for Kawaguchi-1, (c) 135 mg dw L⁻¹ d⁻¹ for Yamanaka, (d) 60 mg dw L⁻¹ d⁻¹ for UTEX 2441, (e) 110 mg dw L⁻¹ d⁻¹ for UTEX LB572, and (f) 195 mg dw L⁻¹ d⁻¹ for Botryococcus sudeticus (UTEX 2629)

Fig. 3

Microscopic observations of a dispersed B. braunii var. Showa micro-colony, showing the grape-seed-like green cells for all B. braunii strains (a-e) and round green cells (f) Botryococcus sudeticus (UTEX 2629). Bars indicate 10 μm

Fig. 4

In vivo buoyant densities of various live Botryococcus cells, sorted according to increasing buoyant density of the samples. (a) Botryococcus braunii var. Showa, (b) Kawaguchi-1, (c) Yamanaka, (d) UTEX 2441, (e) UTEX LB572, and (f) Botryococcus sudeticus (UTEX 2629). A 10–80% (w/v) sucrose gradient was employed with a 10% increment among the gradient steps

Fig. 5

Aqueous buoyant separation of extracellular hydrocarbons from the Botryococcus biomass following sonication of (a) Botryococcus braunii var. Showa, and (b) Botryococcus braunii Kawaguchi-1. A 10–80% (w/v) sucrose gradient was employed with a 10% increment among the gradient steps

Fig. 6

Absorbance spectra of heptane extracts of Botryococcus braunii var. Showa (a and c), and Botryococcus braunii Kawaguchi-1 (b and d) micro-colonies. Absorbance of extracts in the blue (380–520 nm) region of the spectrum (a and b) are attributed to extracellular carotenoids from the two strains. Absorbance of extracts in the far UV (190–220 nm) region of the spectrum (c and d) are attributed to extracellular botryococcenes from the two strains, respectively