NREL Research Technician Nick Sweeney inoculates algae being grown in a tent reactor. Algae brought back to active states from cryogenic tanks need aerobic environments to meet their full potential as biofuels. Photo by Dennis Schroeder, NREL

NREL Research Technician Nick Sweeney inoculates algae being grown in a tent reactor. Algae brought back to active states from cryogenic tanks need aerobic environments to meet their full potential as biofuels. Photo by Dennis Schroeder, NREL

At the Energy Department’s National Renewable Energy Laboratory (NREL), and at dozens of other labs nationwide, cryogenically stored algae emerge from their frigid and foggy environment to live again. Awakening a dormant algae sample, though, takes precision and care.

At NREL, that task often falls to Research Technician Nick Sweeney. “We do cryogenic resurrection of algae from our library of strains,” he said. “We get calls from other labs, universities, private companies, or from our own researchers requesting a particular strain. It’s not as simple as pulling something frozen out of the freezer. The protocol is to slowly bring it back to a state where its biology is going to start functioning normally.”

An NREL researcher removes boxes of algae cultures from a cryopreservation tank. The tank is cooled using liquid nitrogen, which freezes and preserves the algae cultures. NREL researchers are studying algae as a potential source of biofuels. Photo by Pat Corkery

An NREL researcher removes boxes of algae cultures from a cryopreservation tank. The tank is cooled using liquid nitrogen, which freezes and preserves the algae cultures. NREL researchers are studying algae as a potential source of biofuels. Photo by Pat Corkery

The frozen algae samples are held in 2-milliliter vials in a 4-foot-high cryogenic tank, some frozen for as long as five years. The cryogenic tank at NREL is set at -195°C. Liquid nitrogen provides the chill for the tank, which has a kind of ‘Lazy Susan’ inside to make it easier to access the 500-plus strains. The algae stay alive, but dormant, despite the extreme cold thanks to a 5% solution of a cryogenic protectant solvent such as methanol. It’s totally dark inside the tank. Upon removing a vial from the tank, the cells inside the vial are pelleted and look like coffee grounds.

The first step is to separate the liquid from the solids, using a centrifuge to spin the pellets rapidly. When the water, salts, and cryogenic protectant solvents are removed, what remains are the green algae cells.

When reviving the cells, it is essential to keep them in the dark for the first several steps of the process. A paper towel around the vial can do the trick.

The aim is to slowly replicate the algae’s natural environment, so a growth medium is introduced. Depending on where the strain came from, the growth medium could be brackish, salty, or mostly fresh water, or it could include numerous ingredients that replicate the natural water chemistry.

The samples are next put in a shaker to make sure the cells mix well with the growth medium. After the pellets are re-suspended in the growth medium, they’re placed in a subdued lighting environment for 24 hours. They can be damaged if they’re immediately hit with full light. Gradually, the light intensity is increased. The algae replicate on their own, and soon the population has doubled, quadrupled, and more.

—Bill Scanlon