Multi-pronged approach to improving carbon utilization by cyanobacterial cultures
The goal of this project was to increase the efficiency of carbon utilization by a Synechocystis biofuel production strain in a photobioreactor system by at least 50% using a multi-pronged approach that combined the best features of both biological and physicochemical CO2 capture technologies to: 1) increase rates and extent of CO2 absorption into the culture medium by addition of biocompatible amine solvents and a nanobubble gas delivery system, and 2) increase cellular rates of inorganic carbon uptake and carbon fixation through genetic engineering. In addition, the project plan included evaluating the use of fermentation effluent gases as a CO2 source and demonstrating performance under outdoor conditions in closed-pond photobioreactor systems. These combined efforts would be guided by integrated technoeconomic and life-cycle analyses. Both physicochemical CO2 capture technologies explored proved to be successful. The amine solvent monoethanolamine (MEA) was found to be biocompatible with a Synechocystis laurate production strain. Using an adaptive evolution strategy, an MEA-tolerant laurate production strain was developed that showed significantly increased carbon utilization over the parent strain. To our knowledge this was the first time nanobubble (NB) technology had been used as a carbon delivery system for algae production and it also proved quite successful as a proof of concept. Solutions containing CO2 nanobubbles (Nano-BG11) at concentrations significantly higher than the aqueous CO2 concentrations achieved with gas sparging were successfully generated and characterized with regard to bubble size (nm), concentration (NB/L), and pH stability as per project plan. Growth studies proved challenging. However, once the source of a puzzling growth defect was found and remedied Synechocystis was able to grow somewhat better in Nano-BG11 than in standard BG11 medium. Several biological approaches were used to genetically engineer Synechocystis to increase carbon utilization efficiency including: 1) overexpression of multiple homologs of the low-flux/high-affinity bicarbonate (HCO3-) transporter BicA to increase carbon uptake (Kamennaya et al., 2015; Gupta et al., 2020), 2) overexpression of the native bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (BiBPase) gene to increase the rate of regeneration of ribulose-1,5- bisphosphate (RuBP) (Rosgaard et al., 2012), 3) introduction of a synthetic C4 cycle to enable fixation of CO2 at night and release of CO2 during the day when the Calvin-Benson-Bassham (CBB) cycle is active, and 4) incorporation of a synthetic malyl-CoA-glycerate (MCG) pathway in an effort to provide a simple and cost-effective mechanism for maximizing CO2 fixation. These approaches yielded mixed results as was anticipated: not all new experimental approaches can be expected to be successful. The biofuel production strains were not able to stably express the MCG pathway in its current arrangement and bicA overexpression did not improve growth or carbon uptake. The C4 cycle was successfully expressed but did not yield a growth or production benefit. While BiBPase overexpression was successful, there was no significant growth advantage. However, the BiBPase overexpression strain was used to successfully complete the adaptive laboratory evolution (ALE) experiment yielding a faster growing and more productive strain as compared to the precursor strain. Outdoor cultivation trials were more challenging than expected but were quite fruitful. The baseline cultivation run was the first successful completion of a 30-day outdoor trial with multiple resets of a Synechocystis biofuel-producing strain and set the baseline performance criterion for further testing. The comprehensive cultivation trials were also successfully completed. However, due to the challenges in growing cultures in Nano-BG11 at laboratory scale, a decision was made at the Interim Verification that the outdoor trial of the nanobubble delivery system would be suspended. Concurrent Techno Economic Analysis (TEA) and Life Cycle Analysis (LCA) were performed on a cyanobacteria-to-fuels facility with secreted products. The biorefinery model developed for a Synechocystis sp. PCC 6803 methyl laurate production strain included all aspects of cultivation, separation of the secreted methyl laurate, biomass harvesting and fuel processing via hydrothermal liquefaction (HTL) of the dewatered biomass. The assessments leverage Monte Carlo analysis (MCA) to address uncertainty and variability inherent in the most significant input parameters, replacing them with probabilistic functions. In initial modeling, the Nano Gas system was evaluated and shown to not be competitive with standard sparging. However, the final updated GWP, which incorporated changes to both the Nano Gas delivery and methyl laurate harvesting systems achieved a GWP that surpasses the Renewable Fuel Standard.
- Research Organization:
- Arizona State Univ., Tempe, AZ (United States)
- Sponsoring Organization:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE)
- DOE Contract Number:
- EE0008515
- OSTI ID:
- 1877928
- Report Number(s):
- DOE-ASU-08515
- Country of Publication:
- United States
- Language:
- English
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