This Small Business Innovation Research Phase II project develops novel technologies for separation and concentration of intrinsically magnetically susceptible algae for production of biofuels and biochemicals. Phase II builds on the feasibility demonstrated in Phase I using a model alga. During Phase II, an algal strain used for production of renewable biofuel feedstock will be utilized. Novel transformation vectors and tools developed for a production strain, Auxenochlorella protothecoides, will be used to make the algae magnetically susceptible. These traits provided an advantage vs. wild-type strains in growth in low iron medium for the model alga. Phase II will test modified algal strains at lab- and subpilot-scale to determine their performance in growth, and competition with wild-type and weedy algal strains. Additionally, strains will be tested for their ability to be separated or harvested magnetically. This separation will be modeled to determine cost efficacy for primary or secondary dewatering. The specificity of this separation will also be evaluated in relation to downstream use in a heterotrophic bioreactor. The OSU collaboration allows use of these strains in novel rare earth magnetic separators. The endpoint will be novel technologies to improve the overall cost structure for the production of algae-derived biofuels and biochemicals.
The broader impact/commercial potential of this Phase 2 research project will be to provide improvements in the economics of producing renewable biofuels using algae as the production system. It directly addresses one of the major issues with algal biofuels, cost effective dewatering. It also provides a potential selective advantage of the modified strains by improving its ability to compete for iron in an open environment (such as open raceways or photobioreactors). The nation has a critical need to improve its energy security and reduce its dependence on fossil fuels. This research will help address both of these needs. The overall purpose of this research project is business related and focused on commercialization of this technology through integration in a biofuel production process. This research project focuses on a high cost portion of the production process, dewatering, as well as a critical unit process, the heterotrophic bioreactor. The collaboration with OSU and the Cleveland Clinic will result in training of students in this area. The company plans to publish the results of this project once proper control of the intellectual property generated is accomplished.
Phycal, Inc. is developing a two stage process for algal biofuels production with KRT1006, a strain of Auxenochlorella protothecoides, Stage I is the photoautotrophic growth of algae in open ponds using solar energy to drive the assimilation of carbon dioxide from industrial emissions and nutrients partially sourced from wastewater into algal biomass as fast as possible. This biomass is then concentrated by ~100x then transferred to large heterotrophic bioreactors for Stage II. Stage II (called the Heteroboost process) involves the biotransformation of fixed carbon (e.g. glucose) into lipid. A biotransformation process is one in which growth of the organism is uncoupled from the product formation thereby enabling a higher efficiency of conversion of substrate to product. During the Heteroboost process, significant lipid accumulation is observed with minimal additional non-lipid biomass produced. Therefore, the Phycal production process spatially and temporally separates structural biomass accumulation (pond mass culture) from the majority of lipid production and accumulation (Heteroboost process bioreactors). To enable an efficient biotransformation reaction capable of producing oils on a scale sufficient to replace commodity fuels, it is necessary to develop strategies to produce large amounts of algal biomass with low contaminants but at high algal cell density (~50 g/L). Open pond cultivation is generally regarded as the least expensive method of algal biomass generation, although not the only method possible. Contamination control is of significant importance to open pond cultivation. Contaminating weedy species of algae, bacteria, fungi, and protists can absorb light, consume nutrients, and attack productive algae. Such contamination diminishes pond productivity and significantly reduces the efficiency of downstream processing. In the case of the Heteroboost process, the presence of contaminating bacteria can have a significantly negative impact on productivity and substrate costs. To address these concerns, this research project attempted to develop strains (with high iron scavenging and storage capabilities) along with related growth and harvesting technologies that reduce contamination levels as the biomass enters into the Heteroboost process. Strains with enhanced iron homeostasis may enable two advantageous processes. The first decreases the bioavailable iron in the medium. Low iron availability reduced growth by contaminating species. Productive algae that have enhance iron uptake and storage can, at high density, scavenge iron to very low levels. Such activity reduces the amount of available iron to contaminants thereby contributing to their growth inhibition. An interesting by-product of this improved uptake and storage of iron is an enhanced magnetic susceptibility of the algae. This intrinsic magnetic susceptibility presents the potential for use of magnetic separation technology to not only separate the productive species from the contaminating species but also reducing the energy requirements for concentrating the algae from 0.7 g/L in open ponds to 50 g/L, the input concentration of the Heteroboost process. Three target genes were identified which are involved in mediating iron homeostasis. The Fea1, protein is a high affinity Fe2+ transporter native to Chlamydomonas reinhardtii that is well characterized and is known to selectively transport iron. This reduces the risk of accumulating other heavy metals which may become toxic to the algae. An associated Fre1 protein acts as an iron reductase (Fe3+ to Fe2+) that is also membrane associated. It is suggested that Fre1 reduces iron so that it is available for transport by Fea1. Both of their respective genes are known to be induced under low iron conditions. The third target gene encodes the Fer1 or ferritin protein containing twenty four identical ferritin subunits. Fea1, Fer1, and Fre1 genes that enhance cellular iron accumulation were successfully inserted into Phycal's KRT1006 biofuels production strain; however, characterizing the improved phenotypes yielded mixed results. Cell tracking velocimetry analysis indicated that the mean magnetic mobility of the genetically modified strains were higher than the wild type or unmodified strain. The data from the cultivation experiments suggested that the genetically modified strains grew, up took iron, and accumulated a similar amount of iron as the wild type strain. All strains up took iron at a similar rate during the first 3 days of growth then the uptake rate was reduced, and all strains accumulated higher iron content when grown in a medium with higher iron. Magnetic deposition microscopy separation results revealed that approximately 10% more of the genetically modified strains separated from high iron medium compared to the wild type strain, but the reproduced cell tracking velocimetry results were inconclusive, being within the error of the instrument. There is evidence that these GMO strains possess enhanced magnetic properties, but not enough to make an immediate impact on the integrated algal biofuel production system economics. The creation of Fer1/Fea1 and Fre1 constructs was a significant accomplishment during this project and will be used in future projects.
