Arizona State researchers engineer fatty-acid secreting cyanobacteria for biofuel production; Green Recovery method for fatty acid recovery from cyanobacterial biomass
In two open-access papers published in the Proceedings of the National Academies (PNAS), a team from Arizona State University describes the genetic modification of cynanobacteria to produce and secrete fatty acids for renewable biofuel production, as well as a Green Recovery strategy to convert cyanobacterial membrane lipids into fatty acids.
The Green Recovery strategy can be incorporated into the fatty-acid-secretion strains, enabling fatty acid recovery from the remaining cyanobacterial biomass that will be generated during fatty acid biofuel production in photobioreactors.
Although cyanobacteria usually do not accumulate neutral lipids, their photosynthetic membranes are made of diacylglyerol lipids and they have a robust lipid biosynthetic metabolism. Additionally, cyanobacteria are much more genetically manipulatable than algae.
In the field of algal or cyanobacterial fuels, biomass serves as a criterion for their potential biofuel productivity. Typically, cells with lipids need to be harvested, dried, and then extracted by solvents. Biomass extraction processes are energy intensive, usually accounting for 70–80% of the total cost of biofuel production. To skip these steps, we genetically engineered cyanobacteria to continuously secrete free fatty acids (FFA), which can be directly collected from the culture medium. In this scheme, the cyanobacteria are not the biomass that must be processed; they are cell factories that convert solar energy and CO2 into biofuel precursors.—Liu et al. (2011a)
The ASU team made six successive generations of genetic modifications of the cyanobacterium Synechocystis sp. PCC6803 wild type (SD100), and increased the fatty acid secretion yield to 197 ± 14 mg/L of culture in one improved strain at a cell density of 1.0 × 109 cells/mL by adding codon-optimized thioesterase genes and weakening polar cell wall layers.
Although these strains exhibited damaged cell membranes at low cell densities, they grew more rapidly at high cell densities in late exponential and stationary phase and exhibited less cell damage than cells in wild-type cultures.
We found both disadvantages and advantages of FFA overproduction on growth of SD strains. The disadvantage was the fragility of SD cells with CO2 aeration at low cell density, which caused a long lag phase for FFA-secreting SD cultures. In addition, surface layer elimination contributed to cell fragility. Proper cell density is therefore important for SD cultures with multiple gene alterations to grow in a healthy manner with added CO2 aeration as described in SI Text. However, the increased cell fragility may cause growth problems and low practical yields under industrial conditions.
We believe that the industrial working strains need hyper and endurant biofuel productivity, robust cell growth, and cell rigidity. Based on the lessons we learned from the pilot strains, we will avoid modifications that sacrifice cell growth and rigidity in optimizing and reconstructing FFA strains for industrial use.—Liu et al. (2011a)
Green Recovery. Green Recovery takes a different approach to extracting lipids from cynanobacteria, utilizing lipolytic enzymes under the control of promoters induced by CO2 limitation.
...the traditional downstream recovery of microbial lipids, requiring physical cell lysis followed by chemical solvent extraction, accounts for 70–80% of the total cost of biofuel production. We propose a more cost-effective way to harvest lipids from cyanobacterial biomass for biofuel production that precludes the need for mechanically disrupting the cells. To this end, we developed a Green Recovery system in which lipolytic enzymes degrade the membrane lipids into free fatty acids (FFA) with the collapse of cells. The Green Recovery system controls the synthesis of lipolytic enzymes using CO2-limitation-inducible promoters, which induce expression of the lipolytic genes upon cessation of CO2 aeration.—Liu et al. (2011b)
The team’s data indicate that strains of the cyanobacterium Synechocystis sp. PCC6803 engineered for Green Recovery underwent degradation of membrane diacylglycerols upon CO2 limitation, leading to release of fatty acids into the culture medium. Recovered fatty acid yields of 36.1 × 10-12 mg/cell were measured in one of the engineered strains (SD239).
The Green Recovery method offers a number of advantages, the researchers suggested, including:
Not requiring traditional biomass processes such as cell harvesting, dewatering, cell disruption, solvent extraction, or inducer molecules considerably reduces the cost of lipid recovery.
Because continuous agitation is not required for Green Recovery, this system only needs sunlight and possibly intermittent agitation to convert biomass into FFAs.
Lipolytic enzymes convert diacylglycerols in the membranes into FFAs, which, due to their low density and low solubility in water, are easier to harvest and refine than the diacylglycerol lipids.
Some unsaturated species can be saturated by hydrogenation if the FFA products will undergo a hydrogen decarboxylation to yield alkanes.
Green Recovery with FFA Secretion. ASU researchers also incorporated the Green Recovery system into the FFA-secretion strains described above. They found that FA-secretion strains that harbor the Green Recovery system are still able to release membrane lipids as FFAs at rates faster than non-FFA secretion strains following CO2 limitation.
They found that the FFAs recovered from overproducing strains are highly saturated and rich in C12:0 and C14:0, whereas FFAs obtained via the Green Recovery system contained substantial amounts of unsaturated fatty acids and only a small portion of C12:0 and C14:0, which is the same composition observed in membrane lipids. The released FFA amount from membrane lipids was similar to the amount of secreted FFA. Thus, the FFAs recovered from the combination strains after CO2 limitation was a mixture of the overproduced (secreted) FFAs and the released membrane FFAs from Green Recovery.
Green Recovery exhibits other advantages when combined with the previously described cyanobacterial FFA-secretion system. The FFA-secretion system avoids the energy-intensive biomass processes such as concentration and extraction by directly recovering the secreted FFA from the culture medium. However, the FFA-secretion system still requires substantial biomass to achieve cost-effective FFA production, which means a significant amount of fixed carbon has to be converted and stored as lipid membranes.
It is expected that the Green Recovery system will recover the membrane lipids in the potential remaining cyanobacterial biomass generated by the FFA-secretion system, and will also cause cell lysis and release of the unsecreted intracellular FFAs. To our surprise, incorporation of the Green Recovery system into the FFA-secretion strains resulted in an increased damage rate upon CO2 limitation. We postulate that secretion of FFAs through the cytoplamstic membranes creates some lesions in the membranes that facilitate the contact of lipolytic enzymes to the acyl glycerol ester bonds.
These findings demonstrate the practical combination of the FFA-secretion system and Green Recovery in photobioreactors, where the old cultures or the spent biomass can be utilized for extra FFA yield. This Green Recovery is also applicable in the SD100 strains we generated for producing other biofuel molecules, such as triacylglycerols, fatty acyl methyl, or ethyl esters (biodiesel), and alkanes. However, the yields of the other biofuel molecules are lower than would be needed for a productive process. We believe that cyanobacterial biofuel will be instrumental in developing a carbon neutral source of sustainable fuels.—Liu et al. (2011b)
Xinyao Liu, Jie Sheng, and Roy Curtiss III (2011a) Fatty acid production in genetically modified cyanobacteria. PNAS doi: 10.1073/pnas.1103014108
Xinyao Liu, Sarah Fallon, Jie Sheng, and Roy Curtiss III (2011b) CO2-limitation-inducible Green Recovery of fatty acids from cyanobacterial biomass. PNAS doi: 10.1073/pnas.1103016108