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  1. Background: Although microalgal biofuels have potential advantages over conventional fossil fuels, high production costs limit their application in the market. We developed bio-flocculation and incubation methods for the marine alga Nannochloropsis oceanica CCMP1779 and the oleaginous fungus Mortierella elongata AG77 resulting in increased oil productivity. Results: Grown separately and then combining the cells the M. elongata mycelium could efficiently capture N. oceanica due to an intricate cellular interaction between the two species leading to bio-flocculation. Use of a high-salt culture medium induced accumulation of triacylglycerol (TAG) and enhanced the content of polyunsaturated fatty acids (PUFAs) including arachidonic acid (ARA) andmore »docosahexaenoic acid (DHA) in M. elongata. To increase TAG productivity in the alga, we developed an effective reduced nitrogen supply regime based on ammonium in environmental photobioreactors (ePBRs). Under optimized conditions, N. oceanica produced high levels of TAG that could be indirectly monitored by following chlorophyll content. Combining N. oceanica and M. elongata to initiate bio-flocculation yielded high levels of TAG and total fatty acids, ~15% and 22% of total dry weight (DW), respectively, as well as high levels of PUFAs. Genetic engineering N. oceanica for higher TAG content in nutrient-replete medium was accomplished by overexpressing DGTT5, a gene encoding the type II acyl-CoA:diacylglycerol acyltransferase 5. Combined with bioflocculation this approach led to increased production of TAG under nutrient replete conditions (~10% of DW) compared to the wild type (~6% of DW). Conclusions: The combined use of M. elongata and N. oceanica with available genomes and genetic engineering tools for both species opens up new avenues to improve bio-fuel productivity and allows the engineering of polyunsaturated fatty acids. Keywords: Microalgae, Filamentous fungi, Flocculation, Cell wall interaction, Biofuel, Nitrogen starvation, Polyunsaturated fatty acid, Triacylglycerol« less
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