Optimized inorganic carbon regime for enhanced growth and lipid accumulation in Chlorella vulgaris

Abstract

Background: Large-scale algal biofuel production has been limited, among other factors, by the availability of inorganic carbon in the culture medium at concentrations higher than achievable with atmospheric CO2. Life cycle analyses have concluded that costs associated with supplying CO2 to algal cultures are significant contributors to the overall energy consumption.

Results: A two-phase optimal growth and lipid accumulation scenario is presented, which (1) enhances the growth rate and (2) the triacylglyceride (TAG) accumulation rate in the oleaginous Chlorophyte Chlorella vulgaris strain UTEX 395, by growing the organism in the presence of low concentrations of NaHCO3 (5 mM) and controlling the pH of the system with a periodic gas sparge of 5 % CO2 (v/v). Once cultures reached the desired cell densities, which can be "fine-tuned" based on initial nutrient concentrations, cultures were switched to a lipid accumulation metabolism through the addition of 50 mM NaHCO3. This two-phase approach increased the specific growth rate of C. vulgaris by 69 % compared to cultures sparged continuously with 5 % CO2 (v/v); further, biomass productivity (g L(-1) day(-1)) was increased by 27 %. Total biodiesel potential [assessed as total fatty acid methyl ester (FAME) produced] was increased from 53.3 to 61 % (FAME biomass(-1)) under the optimized conditions; biodiesel productivity (g FAME L(-1) day(-1)) was increased by 7.7 %. A bicarbonate salt screen revealed that American Chemical Society (ACS) and industrial grade NaHCO3 induced the highest TAG accumulation (% w/w), whereas Na2CO3 did not induce significant TAG accumulation. NH4HCO3 had a negative effect on cell health presumably due to ammonia toxicity. The raw, unrefined form of trona, NaHCO3∙Na2CO3 (sodium sesquicarbonate) induced TAG accumulation, albeit to a slightly lower extent than the more refined forms of sodium bicarbonate.

Conclusions: The strategic addition of sodium bicarbonate was found to enhance growth and lipid accumulation rates in cultures of C. vulgaris, when compared to traditional culturing strategies, which rely on continuously sparging algal cultures with elevated concentrations of CO2(g). This work presents a two-phased, improved photoautotrophic growth and lipid accumulation approach, which may result in an overall increase in algal biofuel productivity.

Keywords: Bicarbonate; Biodiesel; CO2; Chlorella vulgaris; Fatty acid methyl ester (FAME); Microalgae; Nitrogen limitation; Triacylglycerol (TAG).

Fig. 1

Extractable lipid class and FAME profiles for cultures of C. vulgaris re-suspended into medium depleted of nitrogen and supplemented with various bicarbonate salts. Final concentrations of bicarbonate salts per experimental condition: 0 mM HCO₃ ⁻ (control), 50 mM ACS grade NaHCO₃ (grade 1), 50 mM industrial grade NaHCO₃ (grade 2), 50 mM KHCO₃, 50 mM Na₂CO₃, and 25 mM NaHCO₃∙Na₂CO₃ (25 mM of sesquicarbonate was used to provide equimolar carbon). Values are reported for the completion of the experiment (n = 3). All values expressed as weight percent (% weight extractable lipid or weight FAME/weight biomass)

Fig. 2

pH for cultures of C. vulgaris re-suspended into medium depleted of nitrogen and supplemented with various bicarbonate salts. Final concentrations of bicarbonate salts per experimental condition: 0 mM HCO₃ ⁻ (control), 50 mM ACS grade NaHCO₃ (grade 1), 50 mM industrial grade NaHCO₃ (grade 2), 50 mM KHCO₃, 50 mM NH₄HCO₃, 50 mM Na₂CO₃, and 25 mM NaHCO₃∙Na₂CO₃ (25 mM of sesquicarbonate was used to provide equimolar carbon) (n = 3)

Fig. 3

Growth (cells mL⁻¹) of cultures of C. vulgaris cultured under various inorganic carbon regimes. (Square) Continuous sparge of atmospheric air, (triangle) continuous sparge of atmospheric air and supplemented with 5 mM NaHCO₃ at inoculation, (circle) continuous sparge of atmospheric air supplemented periodically with 5 % CO₂ (v/v) to maintain pH between 8.4 and 8.7, (diamond) continuous sparge of atmospheric air supplemented with 5 % CO₂ (v/v) during daytime hours, and (right pointing triangle) the optimized scenario of a continuous sparge of atmospheric air supplemented periodically with 5 % CO₂ (v/v) to maintain pH between 8.4 and 8.7 and an initial addition of 5 mM NaHCO₃ at inoculation plus an additional 50 mM NaHCO₃ just prior to nitrogen depletion to stimulate TAG accumulation (n = 3). Arrow indicates time of 50 mM NaHCO₃ addition just prior to nitrogen depletion of the culture medium

Fig. 4

Total chlorophyll concentration (mg L⁻¹) for cultures of C. vulgaris cultured under various inorganic carbon regimes. (Square) Continuous sparge of atmospheric air, (triangle) continuous sparge of atmospheric air and supplemented with 5 mM NaHCO₃ at inoculation, (circle) continuous sparge of atmospheric air supplemented periodically with 5 % CO₂ (v/v) to maintain pH between 8.4 and 8.7, (diamond) continuous sparge of atmospheric air supplemented with 5 % CO₂ (v/v) during daytime hours, (right pointing triangle) the optimized scenario of a continuous sparge of atmospheric air supplemented periodically with 5 % CO₂ (v/v) to maintain pH between 8.4 and 8.7 and an initial addition of 5 mM NaHCO₃ at inoculation plus an additional 50 mM NaHCO₃ just prior to nitrogen depletion to stimulate TAG accumulation (n = 3)

Fig. 5

Extractable lipid class and FAME profiles for cultures of C. vulgaris when cultured under various inorganic carbon regimes. Experimental conditions: (1) the optimized scenario of a continuous sparge of atmospheric air supplemented periodically with 5 % CO₂ (v/v) to maintain pH between 8.4 and 8.7 and an initial addition of 5 mM NaHCO₃ at inoculation plus an additional 50 mM of ACS grade NaHCO₃ just prior to nitrogen depletion to stimulate TAG accumulation. (2) The same culture conditions as scenario 1 listed above, except TAG accumulation was induced by adding 25 mM NaHCO₃∙Na₂CO₃ (sesquicarbonate). (3) Continuous sparge of atmospheric air supplemented with 5 % CO₂ (v/v) during daytime hours. Values are reported for the completion of the experiment (n = 3). All values expressed as weight percent (% weight extractable lipid or weight FAME/weight biomass)