Enhancement of harvesting efficiency and polyunsaturated fatty acid-rich lipid production of Aurantiochytrium sp. SW1 by co-cultivation with oleaginous fungus Cunninghamella bainieri 2A1

Aims@#Thraustochytrids have been shown to be excellent lipid producers due to their ability to accumulate over 50% lipid (g/g biomass) containing up to 50% docosahexaenoic acid (DHA). However, efficient and cost-effective cell recovery of lipid-rich biomass has become a significant challenge at the industrial scale. In this study, we attempted to enhance the harvesting efficiency (HE) and the DHA content of Aurantiochytrium sp. through co-cultivation with a γ-linolenic acid (GLA)-producing oleaginous filamentous fungus, Cunninghamella bainieri 2A1.@*Methodology and results@#A 72 h old C. bainieri 2A1 culture in the form of loose mycelia or pellets of various sizes was added into 72 h old Aurantiochytrium sp. cultures and further incubated for 48 h. The HE of Aurantiochytrium sp. was then determined by comparing the remaining OD values of the supernatant with and without minimal centrifugation at 4000× g. Results showed that 63.23% of HE was achieved without centrifugation from co-cultivation with dispersed mycelia. Higher HE between 96.71-99.55% was achieved when centrifugation was implemented, with the highest value resulting from co-cultivation with dispersed mycelia. These are higher than HE of centrifuged control cultures (80%) consisting of Aurantiochytrium sp. monocultures, suggesting that co-cultivation with C. bainieri 2A1 facilitates the recovery of Aurantiochytrium sp. cells. Moreover, the co-cultivation also resulted in a 28% increase in DHA compared to non-optimized cultures.@*Conclusion, significance and impact of study@#This study provides the first evidence of enhancement in harvesting and DHA content of oleaginous thraustochytrids that could be achieved through co-cultivation with oleaginous fungi.

Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation

Baffled shake flask cultivation of Aurantiochytrium sp. B-072 was carried out at in a glucose-monosodium glutamate mineral medium at different C/N-ratios (30-165) with glucose fixed at 90 g/L. With increasing C/N-ratio, a modest increase in lipid content (60 to 73 % w/w) was observed whereas fat-free biomass decreased but overall biomass showed little variation. FA-profiles were not affected to a large extent by C/N-ratio and absolute docosahexaenoic (DHA)-levels fell in narrow range (5-6 g/L). However at C/N > 64 a rapid decrease in lipid synthetic rate and/or incomplete glucose utilization occurred. Glucose and FA-fluxes based on fat-free biomass peaked at a C/N ratio of 56. This condition was chosen for calculation of the redox balance (NAD(P)H) and energy (ATP) requirement and to estimate the in vivo P/O ratio during the main period of fatty acid biosynthesis. Several models with different routes for NADPH, acetyl-CoA formation and re-oxidation of OAA formed via ATP-citrate lyase were considered as these influence the redox- and energy balance. As an example, using a commonly shown scheme whereby NADPH is supplied by a cytosolic "transhydrogenase cycle" (pyruvate-OAA-malate-pyruvate) and OAA formed by ATP-citrate lyase is recycled via import into the mitochondria as malate, the calculated NADPH-requirement amounted to 5.5 with an ATP-demand of 10.5 mmol/(g fat-free biomass x h) and an in vivo P/O-ratio (not including non-growth associated maintenance) of 1.6. The lowest ATP requirement is found when acetyl-CoA would be transported directly from the mitochondria to the cytosol by carnitine acetyltransferase. Assay of some enzymes critical for NADPH supply indicates that activity of glucose-6-phosphate dehydrogenase, the first enzyme in the HMP pathway, is far insufficient for the required NADPH-flux and malic enzyme must be a major source. Activity of the latter (ca. 300 mU/mg protein) far exceeds that in oleaginous fungi and yeast.

Growth, fatty acid profile in major lipid classes and lipid fluidity of Aurantiochytrium mangrovei Sk-02 as a function of growth temperature

Aurantiochytrium mangrovei Sk-02 was grown in a medium containing glucose (40 g/l), yeast extract (10 g/L) and sea salts (15 g/L) at temperatures ranging from 12 to 35°C. The fastest growth (µmax= 0.15 h-1) and highest fatty acid content of 415 mg/g-dry cell weight were found in the cells grown at 30°C. However, the cells grown at 12°C showed the highest percentage of polyunsaturated fatty acid (PUFA) (48.6% of total fatty acid). The percentage of docosahexaenoic acid (DHA) and pentadecanoic acid (C15:0) decreased with an increase in the growth temperature, whereas, palmitic acid (C16:0), stearic acid (C18:0) and DPA (C22:5n6) increased with an increase in the growth temperature. The composition of the major lipid class (%w/w) was slightly affected by the growth temperature. The fluidity of the organelle membrane or intracellular lipid (by DPH measurement) decreased with an increase in the growth temperatures, while the plasma membrane fluidity (by TMA-DPH measurement) could still maintain its fluidity in a wide range of temperatures (15 - 37°C). Furthermore, the distribution of DHA was found to be higher (36 - 54%) in phospholipid (PL) as compared to neutral lipid (NL) (20 - 41%).