1. Safe use and sampling of a photobioreactor sparged with corrosive gases
NOTE: This method does not describe appropriate procedures for safe sampling of microalgal cultures that produce or consume highly flammable gases.
2. Preparation of the microalgal inoculum
3. Setup and operation of photobioreactor
4. Adapting the photobioreactor and experimental setup for toxic gas use
CAUTION: The corrosive gases in real or simulated flue gas are corrosive and toxic. These gases pose serious risk if inhaled.
NOTE: This method does not describe appropriate materials for safe cultivation of microbes that produce or consume highly flammable gases (i.e., methane, hydrogen, etc.).
5. Measuring microalgal biomass productivity
6. Biomass productivity modeling and rate calculations
A calibration curve for the green microalgae, S. obliquus, harvested in the exponential phase, was established with OD750 and dried biomass concentrations (Figure 2). The linear regression had an R2 value of 0.9996.
An S. obliquus culture was started in a 250 mL Erlenmeyer flask from a culture stored on a refrigerated agar plate. The microalga was inoculated in 3N-BBM with 10 mM HEPES buffer and sparged with 2.2% CO2 in a 2 L photobioreactor with 1.5 L working volume (0.07 vvm) (Figure 1). The batch was tracked via OD750; the biomass concentrations were calculated from the calibration curve, and then modeled with a logistic curve (Figure 3). The photobioreactor maintained the culture at pH 6.8, 100 cm3 min-1 total gas flow rate, continuous 280 μmol m-2 s-1 illumination, and 27 °C. The logistic curve fit biomass concentration data from lag to exponential to stationary phase. From the logistic model, the maximum biomass concentration during the batch was 2070 ± 20 mg L-1, maximum biomass productivity occurred at 4.6 day, and the rate of specific biomass productivity was 1.0 d-1. The maximum biomass productivity, calculated from the derivative of the logistic curve at the time of maximum growth, was 532 ± 60 mg L-1 d-1.
The well-mixed room model was used to calculate the accumulated concentration of NO2, SO2, and CO in the case of fume hood failure for 24 h. These values were compared to the exposure limits (Table 2). For example, in the scenario where 0.05 L min-1 of 400 ppm NO2 is released during a fume hood failure period of 24 h, the well-mixed room model with inputs of calculated G = 0.0377 mg min-1, Q = 0.0001 m3 min-1, V = 100 m3, and maximum time for simulation = 1440 min predicts NO2 accumulation to 0.54 mg m-3 (0.29 ppm), which is above the acceptable chronic exposure limit (American Conference of Governmental Industrial Hygienists threshold limit value [ACGIH TLV]) and below the short-term exposure limit (STEL).
A promising preliminary trial with simulated flue gas achieved a greater maximum microalgal biomass productivity rate (690 ± 70 mg L-1 d-1) than that of 12% CO2 and ultra-zero air (510 ± 40 mg L-1 d-1) (Figure 4). Prior to the experiment, a gas monitor was calibrated with CO, NO2, and SO2. The simulated flue gas experiment was carried out without any risk to personnel or damage to equipment from corrosive gases.
Figure 1: Bench-top photobioreactor illuminated by red and blue LED lights. The photobioreactor operates as a 2 L batch reactor with 1.5 L working volume. The photobioreactor is continuously fed with gases through the sparging ring and excess gas vents through ports in the headplate. Adapted with permission from Molitor et al.5. Please click here to view a larger version of this figure.
Figure 2: Calibration curve relating OD750 a S. obliquus cell dry weight. S. obliquus cell culture light absorption was measured at 750 nm, then cells were filtered and dried to obtain cell dry weight measurements. Reprinted with permission from Molitor et al.5. Please click here to view a larger version of this figure.
Figure 3: S. obliquus growth data at 2.2% CO2 input modeled with a logistic regression. The data points represent biomass values as calculated from optical density measurements. The data have been modeled with a logistic regression through a least squares fit; where L = 1955 mg L-1, k = 1.154 d-1, and x0 = 3.317 d. R2 = 0.995. Please click here to view a larger version of this figure.
Figure 4: Modeled S. obliquus growth at 12% CO2, with and without additional simulated flue gas components. The biomass measurements from each batch of microalgae were modeled with logistic regressions. Please click here to view a larger version of this figure.
Component | Percent |
H2O | 12.6% |
CO2 | 11.6% |
O2 | 5.8% |
CO | 0.048% |
SO2 | 0.045% |
NO2 | 0.022% |
N2 | 69.9% |
Table 1: Composition of coal-fired power plant emissions. These quantities were averaged from the University of Iowa power plant emissions data collected at minute intervals over the span of 10 h.
Toxic gas | TWA | CEILING | STEL | NIOSH IDLH | NIOSH REL | ACGIH TLV | CDC Description |
CO | 35 ppm | 200 ppm | – | 1,200 ppm | 35 ppm | 25 ppm | Colorless, odorless |
SO2 | 2 ppm | 100 ppm | 5 ppm | 100 ppm | 2 ppm | 2 ppm | Colorless gas with a characteristic, irritating, pungent odor |
NO2 | 3 ppm | 5 ppm | 1 ppm | 13 ppm | 1 ppm | 0.2 ppm | Yellowish-brown liquid or reddish-brown gas (above 70 °F) with a pungent, acrid odor |
Table 2: Exposure limits and descriptions for toxic gases (CO, SO2, NO2) in flue gas. OSHA TWA: time weighted average (usually 8 h period), CEILING: value never to be reached, STEL: short-term exposure limit (TWA over 15 min), NIOSH IDLH: danger to life and health, NIOSH REL: 15 min exposure limit, ACGIH TLV: acceptable chronic exposure limit, no ill effects.
Compound | mM |
NaNO3 | 8.82 x 100 |
MgSO4·7H2O | 3.04 x 10-1 |
NaCl | 4.28 x 10-1 |
K2HPO4 | 4.31 x 10-1 |
KH2PO4 | 1.29 x 100 |
CaCl2·2H2O | 1.70 x 10-1 |
ZnSO4·7H2O | 3.07 x 10-2 |
MnCl2·4H2O | 7.28 x 10-3 |
MoO3 | 4.93 x 10-3 |
CuSO4·5H2O | 6.29 x 10-3 |
Co(NO3)2·6H2O | 1.68 x 10-3 |
H3BO3 | 1.85 x 10-1 |
EDTA | 1.71 x 10-1 |
KOH | 5.52 x 10-1 |
FeSO4·7H2O | 1.79 x 10-2 |
H2SO4 (concentrated) | 1 x 10-3 μL |
Table 3: Composition of triple-nitrogen Bold’s basal medium (3N-BBM). The quantity of nitrogen has been tripled from the original Bold’s basal medium11.
Biostat A bioreactor | Sartorius Stedim | 2-liter bioreactor for microbial fermentation; designed to be autoclaved; pH, temperature, gas flow rate control | |
Bump test NO2 gas | Grainger | GAS34L-112-5 | Calibration gas for MultiRAE gas detector |
Bump test O2, CO, LEL gas | Grainger | GAS44ES-301A | Calibration gas for MultiRAE gas detector |
Bump test SO2 gas | Grainger | GAS34L-175-5 | Calibration gas for MultiRAE gas detector |
Corrosion resistant tubing for NO2 gas | Swagelok | SS-XT4TA4TA4-6 | PTFE Core Hose Smooth Bore X Series—Fiber Braid and 304 SS Braid Reinforcement |
Corrosion resistant tubing for SO2 gas | QC Supply | 120325 | Reinforced Braided Natural EVA Tubing – 1/4" ID |
cozIR 100% CO2 meter | Gas Sensing Solutions Ltd. | CM-0121 at CO2meter.com | CO2 meter for concentrations up to 100% |
cozIR 20% CO2 meter | Gas Sensing Solutions Ltd. | CM-0123 at CO2meter.com | CO2 meter for concentrations up to 20% |
Durapore Membrane Filter, 0.45 μm | Millipore Sigma | HVLP04700 | Hydrophilic, plain white, 47 mm diameter, 0.45 μm pore size, PVFD membrane filters |
Gas cylinder regulators | Praxair | PRS 40221331-660 | Single-stage stainless steel regulator configured for 0-15 psi outlet assembly diaphragm valve with 1/4" MNPT threads, Stainless steel to resist corrosion from NOx and SOx |
Gas cylinders | Praxair | Ulta-zero air, high purity CO2, or custom gas composition | Dependent on study objectives |
Gas monitoring and leak detection system | RAE Systems by Honeywell | MAB3000235E020 | Pumped model that detects O2, SO2, NO2, CO, and LEL |
GasLab software | GasLab | v2.0.8.14 | Software for CO2 meter measurements and data logging |
Hose barb | Grainger | Item # 3DTN3 | Used to adapt regulators to tubing, Stainless steel to resist corrosion from NOx and SOx |
K30 1% CO2 meter | Senseair | CM-0024 at CO2meter.com | CO2 meter for concentrations less than 1% |
LED grow panels | Roleadro | HY-MD-D169-S | Red & blue LED light panels |
Memosens dissolved oxygen probe | Endress+ Hauser | COS22D-19M6/0 | Autoclavable (with precautions) dissolved oxygen probe for bioreactor |
Memosens pH probe | Endress+ Hauser | CPS71D-7TB41 | Autoclavable (with precautions) pH probe for bioreactor |
Oven, Isotemp 500 Series | Fisher Scientific | 13246516GAQ | Small oven for drying |
Prism GraphPad software | GraphPad Software | Version 7.03 or 8.0.1 | Graphing software for data organization, data analysis, and publication-quality graphs |
Stem to hose barb fitting | Swagelok | SS-4-HC-A-6MTA | Stainless Steel Hose Connector, 6 mm Tube Adapter, 1/4 in. Hose ID |
Tubing, dilute acid/base transfer | Allied Electronics and Automation | 6678441 | Silicone TP Process Tubing; 1.6mm Bore Size; 3000mm Long; Food Grade |
Tubing, gas transfer | Allied Electronics and Automation | 6678444 | Silicone TP Process Tubing; 3.2mm Bore Size; 3000mm Long; Food Grade |
Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.
Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.
Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.