Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO₂, SO₂, NO₂) from municipal or industrial combustion emissions.

This protocol details the photobioreactor equipment adaptations necessary to cultivate microalgae with corrosive gases and discusses safe operation and sampling of the photobioreactor. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments. This bench-scale system can be used to study the characteristics and productivity of microalgae cultivated with simulated combustion emissions.

This method may be used with other carefully adapted bioreactors or to cultivate other photoautotrophic microorganisms. Visual demonstration of this method is critical because it is a complex protocol that prevents human exposure to the toxic simulated combustion emissions used for microalgae cultivation. To begin, model the possible accumulated concentration of toxic gases in the room if the fume hood were to fail.

Use the American Industrial Hygiene Association's mathematical modeling spreadsheet IH Mod for each gas. From building HVAC maintenance personnel or HVAC technician, obtain Q, the room supply or exhaust air rate in cubic meters per minute. Calculate the volume, V, of the laboratory in cubic meters.

Calculate the contaminant emission rate, G, of each type of toxic gas in milligrams per minute using the equation adapted from the Ideal Gas Law where P is the fraction of pressure exerted by the toxic gas at one ATM, Q gas is the flow rate of the gas in liters per minute, R is the universal gas constant, T is temperature in Kelvin, and MW is the gas'molecular weight in grams per mole. Use the values for V, Q, and G for each gas in the well-mixed room model with option to cease generation and model room purge algorithm in the IH Mod spreadsheet to calculate the accumulated room gas concentrations for each gas over a 24-hour simulation period. Compare these values to the exposure limits.

Set up a toxic gas monitoring system with sensors for each of the toxic gases in use. Calibrate the sensors according to manufacturer's instructions. Bump test frequently.

Locate the gas monitor just outside the fume hood. Prior to the experiment, ensure that all personnel are instructed on the appropriate responses to a toxic gas alarm. Now, prepare 100 milliliters each of one normal sodium hydroxide and one normal hydrochloric acid in two 250 milliliter input solution bottles.

Store metered input solutions in autoclaveable capped bottles equipped with dip tubes and a vent tube with a sterile in-line air filter. Connect the dip tubes to two of the photobioreactor's four input ports using autoclaveable tubing. Insert and screw close the cold finger and exhaust condenser on the photobioreactor head plate.

Insert the inoculation port and screw tightly in place. Add a length of autoclaveable tubing to the section of inoculation port above the photobioreactor head plate. Prior to autoclaving the bioreactor, clamp the tubing closed with an autoclaveable host clamp.

Attach tubing capped with sterile filters to any unused photobioreactor ports. Add 1.5 liters of culture medium. Autoclave the reactor and associated input solutions for 30 to 45 minutes at 121 degrees Celsius.

Pass the 1.6 millimeter inside diameter autoclaveable tubing between the input solutions and their ports through separate peristaltic pumps. Attach the impeller motor to the impeller shaft and tighten the fitting. Arrange LED light panels symmetrically outside the bioreactor according to illumination requirements.

Attach appropriate regulators capable of 20 PSI outlet pressure to the gas cylinders. Attach six millimeter inside diameter pressure-resistant tubing to the regulator outlet hose barb and secure with a hose clamp. Attach the other end of the pressure-resistant tubing to the gas regulating tower gas inlet using a hose barb to a six millimeter stem quick connect fitting secured with a hose clamp.

Connect 3.2 millimeter inside diameter tubing to the gas regulating tower gas outlet using six millimeter quick connect fitting and connect the other end of the outlet tubing to the sparging ring port at the photobioreactor head plate. Set the outlet pressure to 20 PSI on each gas regulator. On the bioreactor interface, set the experimental gas flow rates.

Use the STIRR function to set an impeller rotation rate that is rapid enough for the culture medium to assimilate the sparged gas bubbles. After autoclaving, assemble the photobioreactor and gas cylinders within a walk-in fume hood. Place the photobioreactor on a table inside a secondary container and place gas cylinders in freestanding cylinder colanders or a cylinder rack.

After initiating gas flow, use a wash bottle filled with a 1:100 dilution of dish soap to water to cover the connections between the gas cylinders and bioreactor with a small stream of soap solution. Check for gas leaks indicated by bubbling. When initiating the microalgal experiments, begin sparging of the gas and then adjust pH before inoculation.

Inoculate the photobioreactor by aspirating the prepared microalgal inoculum into a sterile syringe, fitting the syringe to the tubing attached to the inoculation port, opening the inoculation tubing clamp, and depressing the syringe. Check the gas monitor, gas cylinder pressures, and photobioreactor twice daily for elevated levels of toxic gas or indication of leaks. Limit the fume hood sash opening to a width that allows the bioreactor and gas cylinder regulators to be reached.

When sampling, turn the gas cylinder regulators to the closed position to cease gas flow to the reactor. Close the fume hood sash and allow five minutes for the hood to evacuate the corrosive gases. Sample within the fume hood either by opening a head plate port and using a sterile serological pipette or drawing culture into a syringe through the inoculation or sampling port.

In this study, a calibration curve for the green microalgae Scenedesmus obliquus harvested in the exponential phase was established with OD750 measurements and dried biomass concentrations. The biomass concentrations were calculated from the calibration curve and then modeled with a logistic curve where L is the maximum biomass concentration, k is the relative steepness of the exponential phase, x0 is the time of the curve's midpoint, and x is the time. A promising preliminary trial with a simulated flue gas achieved a maximal microalgal biomass productivity rate at 690 milligrams per liter per day which was greater than that of 12%carbon dioxide and ultra-zero air at 510 milligrams per liter per day.

Correct assembly of the system is most important to the procedure for microalgal cultivation and for human safety. The system needs to be constantly monitored with gas sensors. Transfer lines need to be gas tight and the fume hood must be used appropriately.

Pressurized cylinders containing toxic gases are hazardous. Always ensure that the cylinders are secured and only used inside a fume hood after establishing a toxic gas monitoring system.