Growth medium and preparation
The basal growth medium for microalgae culture was a modified BG-11 medium which had the following components (per liter): NaNO3 (100 mg), K2HPO4·3H2O (40 mg), MgSO4·7H2O (75 mg), CaCl2·2H2O (36 mg), citric acid (6 mg), Fe-ammonium citrate (6 mg), EDTA·Na2 (1 mg), NaHCO3 (20 mg), H3BO3 (2.86 mg), MnCl2·4H2O (1.8 mg), ZnSO4·7H2O (0.22 mg), CuSO4·5H2O(0.08 mg), Na2MoO4·2H2O (0.391 mg), and Co(NO3)2·6H2O (0.0494 mg). Sodium nitrate and sodium bicarbonate concentrations were modified as indicated in the text.
The BG-11 medium was prepared with deionized water for laboratory experiments, and with water from Chenghai Lake (containing ~1 g L−1 NaHCO3) for outdoor experiments. The deionized water was sterilized by autoclave, and the water from Chenghai Lake was purified by filtration, and then, the sterile nutrient stock solutions were added to either the deionized water or the water from Chenghai Lake to make final medium. The culture pond was exposed to direct solar irradiation for at least 12 h before medium preparation.
Bubbled column PBR cultivation
Each column of the bubbled column PBR used in this study has an inner diameter of 3 cm and a working volume of 200 mL. The columns were illuminated with white fluorescent tubes for 14 h every day, and the light intensity at the reactor surface was 300 μmol m−2 s−1 (15 mol photons m−2 day−1). CO2-enriched (1 %, v/v) air was injected at the bottom of the column through a glass tube, that is used for CO2 supplement, culture mixing, and O2 exchange. The air flow rate was maintained at 250 mL min−1. A thermostatic water circulator was used as water bath to keep the temperature of the culture columns at 30 °C.
The algal cells were harvested from seed culture and resuspended into sterilized BG-11 medium for inoculation. The cells were grown in batch with an initial cell density of 0.5 ± 0.05 (optical density at 540 nm). To investigate the effects of nitrate and bicarbonate concentration on cell growth and lipid accumulation, different doses of sodium nitrate or sodium bicarbonate were added into each column before inoculation. All of the experiments were carried out in triplicate.
Scale-up cultivation in a 10 L circular pond
A mini circular pond cultivation system was used to simulate the outdoor open pond cultivation under artificial illumination. The circular pond was equipped with four units to control mixing, irradiance, temperature, and pH, as described in previous study [14].
The cells were grown in batch with an initial cell density of 0.1 ± 0.05 (optical density at 540 nm). Total culture volume was about 10 L giving a culture depth (light path) of 10 cm, and the effective illuminated area was 0.1 m2. Banks of white fluorescent tubes were placed above the pond, and provided 300 µmol photons m−2 s−1 with 14 h:10 h light–dark cycle (15 mol photons m−2 day−1). The cultures were kept at 30 °C and mixed at 50 r min−1 continuously. Pure CO2 was dispersed into the culture suspension through a gas diffuser under the programming control of an online pH sensor, to maintain the culture pH within a desired range. In another study, we found that Graesiella sp. WBG-1 was well adapted to culture pH range of 7–10 (unpublished data). Therefore, the culture pH of 9.0 ± 0.5 was maintained in the simulate experiments to facilitate external CO2 uptake. The experiments were carried out in duplicate.
Scale-up cultivation in a 30 L tank PBR
To test the adaptability of the strain to variable environmental conditions, outdoor scale-up cultivations of the Graesiella sp. WBG-1 were carried out in 30 L tank PBR under natural light illumination.
The PBR used in this experiment consisted of four open-top polyethylene tanks placed side-by-side in a single row on a concrete platform. The working volume is 30 L for each culture tank, giving an illuminated area of 0.19 m2. An electromagnetic air compressor continuously blew sterilized air into the culture suspension during cultivation. The air flow was maintained at 3 L min−1. The air was enriched with CO2 (1 %, v/v) during light period to support cell growth and maintain pH within the desired range (9.0 ± 0.5). Natural solar irradiance and environmental temperature were monitored and logged on site with an automatic weather station.
Outdoor cultivation in 200 m2 raceway pond
Pilot-scale evaluation of Graesiella sp. WBG-1 was carried out at Chenghai Lake, Yunnan province, China, (N26°29′29.64″ E100°40′56.12″) using a traditional raceway pond.
The raceway pond was an open system constructed of concrete blocks. The pond was 20 m long, 12 m wide, giving an effective culture area of 200 m2. Two large paddle wheels were installed apart within the raceway for mixing, and were driven by speed-adjustable motors and able to provide a flow velocity of 10–60 cm s−1. The liquid flow velocity was set at 45 cm s−1 in this study, and the paddle wheels were turned on in daytime and turned off in night. Pure CO2 was automatically injected into the culture via a 3 m-long microporous polymer tube (gas diffuser) that was placed at the bottom of the culture pond. The automatic injection of CO2, which was controlled by an online pH sensor, maintained the culture pH within the desired range (pH 9.0 ± 0.5). The working culture depth (light path) was 20 cm, corresponding to 40,000 L culture volume. Natural solar irradiance, environmental temperature, and suspension temperature were monitored and logged on site with an automatic weather station.
Two similar but smaller raceway ponds (20 m2) covered by greenhouse were used for high-quality seed culture preparation. Independent batch cultures in the 200 m2 pond were carried out three times in June 2013, July 2013, and May 2014.
Analytical procedure
Culture growth was estimated by measuring the dry biomass concentration of the culture broth. About 10 mL culture broth was filtered through a pre-dried GF/C glass microfiber filter paper (pore size 0.45 µm), and dried at 105 °C for 4 h, and then weighed to calculate dry biomass concentration (DW, in grams of biomass per liter of culture broth). Daily biomass productivity was calculated by dividing the difference between the DWs at the start time and the end time by its duration (days). The microalga was stained with Nile Red according to Li’s method [10] and observed using a Nikon Eclipse 80i microscope.
A spectrophotometric method, described by Collos et al. [15], was used to monitor residual nitrate concentration. Briefly, the absorbance of culture filtrate (0.22 µm filter) at 220 nm and a pre-constructed standard curve were used to determine residual nitrate.
Cells were collected by centrifugation (5000 rpm for 5 min) and lyophilized (−56 °C cryotrapping, 10–14 Pa vacuum) for biochemical analysis. For lipid quantification, 50 mg of dry algal biomass was fully grounded, transferred to a covered centrifuge tube, and then extracted with a mixture of n-hexane and ethyl acetate (1:1, v:v) for 20 min. The extraction was repeated three times, and all extracts were combined into a pre-weighed glass tube, and then dried under nitrogen protection. The lipids were determined gravimetrically.
Neutral lipid (Triacylglycerides, TAGs) was fractionated from the lipid extracts by column chromatography using a 2 cm × 20 cm column packed with 4 g silica gel 60 [16]. The lipid extracts (~100 mg) were dissolved in 2 mL chloroform and loaded onto the column. TAGs were eluted from the column by 20 mL chloroform. The eluted TAGs fraction were confirmed by TLC, and then dried and quantified gravimetrically.
To analyze fatty acids, about 20 mg of the lipid extract was dissolved in 3 mL n-hexane and then transmethylated by adding 3 mL methanol-KOH (0.5 % KOH) and heating at 50 °C for 60 min. After cooling to room temperature, the hexane layer was separated and dried with anhydrous sodium sulfate. Fatty acid methyl esters were analyzed by gas chromatography (Agilent 7890A) using an HP-5 Phenyl Methyl Siloxan column (30 m × 0.32 mm × 0.25 µm) and a flame ionization detector. 1 µL fatty acid methyl esters solution was injected to the sampler with a splitting ratio of 5:1. The heating program was 150 °C held for 2 min, then increased to 250 °C at a rate of 10 °C per min, and held for 8 min. A standard FAME Mix (Sigma-Aldrich) was used for fatty acid identification.
About 10 mg of lyophilized algal powder was used for total carbohydrate quantification. The algal powder was transferred to a covered tube, fully mixed with 1 mL hydrochloric acid (6 M), to digest at 105 °C for 1 h. After cooling to RT, about 1 mL NaOH (6 M) was added into the solution to neutralize acid, followed by centrifugation at 3500g for 5 min. The supernatant was collected into a new tube and its volume was brought to 2 mL with deionized H2O. 100 µL of the diluted supernatant was diluted again with deionized H2O to 2 mL, mixed with 1 mL phenol (6 %), and then 5 mL sulfuric acid was trickled into the sample for color development. Finally, the optical density at 490 nm was measured on a spectrophotometer. To quantify total carbohydrate content, glucose was used to establish the standard curve.
Protein content was determined as described by Slocombe et al. [17]. Briefly, 10 mg freeze-dried algal powder was suspended in 500 µL of 24 % (w/v) trichloroacetic acid (TCA) and then incubated at 95 °C for 15 min. The lysate containing 24 % (w/v) TCA were cooled to RT and diluted to 6 % (w/v) with 1.5 mL deionized water. The homogenate was centrifuged at 15,000g for 20 min and the supernatant was discarded. The pellets were resuspended in 1 mL NaOH (1 M) by repeated pipetting and then incubated at 40 °C for 2 h. The protein concentration was then spectrophotometrically measured according to standard Bradford assay.
Calculations
Areal biomass concentration (C
biomass, g m−2) was calculated by the following:
$$C_{\text{biomass}} = \frac{{{\text{DW}} \times V}}{S}$$
(1)
with DW as measured biomass dry weight (g L−1), V as culture volume (L), and S as illuminated area (m2).
Biomass productivity (P
biomass, g m−2 day−1) was calculated according to the following:
$$P_{\text{biomass}} = \frac{{C_{{{\text{biomass}},t2}} - C_{{{\text{biomass}},t 1}} }}{t2 - t1 \, }$$
(2)
with C
biomass,t2 and C
biomass,t1 as biomass concentrations at culture time t2 (day) and t1 (day), respectively.
Lipid productivity (P
lipid, g m−2 day−1) was calculated according to the following:
$$P_{\text{lipid}} = \frac{{C_{{{\text{biomass}},t2}} \times C_{{{\text{lipid}},t2}} - C_{{{\text{biomass}},t 1}} \times C_{{{\text{lipid}},t1}} }}{t2 - t1 \, }$$
(3)
with C
lipid,t2 and C
lipid,t1 as lipid content at culture time t2 (day) and t1 (day), respectively.
Average daily light intensity (I
av, mol m−2 day−1) was calculated according to the following:
$$I_{\text{av}} = \frac{{\sum\nolimits_{0}^{t} {I_{\text{inc}} } }}{t \, }$$
(4)
with I
inc as daily incident light intensity (mol m−2 day−1) measured by the automatic weather station.
Biomass-specific light availability (I
biomass, mol g−1 day−1) was calculated according to Ref. [18] by the following:
$$I_{\text{biomass}} = \frac{{I_{\text{av}} }}{{C_{\text{biomass}} \, }}$$
(5)
CO2 utilization rate (R
c, %) was calculated by the following:
$$R_{\text{c}} = \frac{{W_{\text{b}} \times 1000 \times 0.5 \div 12}}{{C_{1} - C_{2} + C_{3} }} \times 100\,\%$$
(6)
with W
b as net increase of biomass (kg) during cultivation; C
1 as dissolved inorganic carbon ([H2CO3] + [CO3
2−] + [HCO3
−], mol) in the medium at the begin of culture; C
2 as dissolved inorganic carbon (mol) in the medium at the end of culture; and C
3 as the total CO2 (mol) used during culture. The bio-fixated carbon (50 % of the biomass is C) divided by consumed carbon was defined as CO2 utilization rate in this study.