Oil extraction report from Chlamydomonas reinhardtii engineered to produce ketocarotenoids

Report of oil extraction efficiency on Chlamydomonas reinhardtii strain engineered to produce ketocarotenoids and astaxanthin. Oil report on lab scale: Oil extraction was performed as described in Perozeni et al. Plant Biotechnology Journal 2020, starting from different volumes of saturated culture (300, 600, 900, and 1800 ml). The cells were pelleted and dissolved in 25 ml of sunflower oil. The extraction was thus performed on wet biomass. After centrifugation, the oil extract was diluted in oil and the absorption spectrum was used to determine the ketocarotenoid concentration as described in Perozeni et al. Plant Biotechnology Journal 2020. The spectra recorded are in the sheet “Raw”. In the sheets “Quantification” there are the results of the ketocarotenoids quantification and the extraction efficiency. In the sheet "summary" the overall results in terms of starting material, final oil volume, ketocarotenoid concentration and extraction efficiency are presented. Samples: 1x: extraction starting from 300ml of C. reinhardtii EN1 strain saturated culture, oil extract diluted 1:10 before measuring the absorption spectrum. 2x: extraction starting from 600ml of C. reinhardtii EN1 strain saturated culture, oil extract diluted 1:100 before measuring the absorption spectrum. 3x: extraction starting from 900ml of C. reinhardtii EN1 strain saturated culture, oil extract diluted 1:100 before measuring the absorption spectrum. 6x: extraction starting from 1800ml of C. reinhardtii EN1 strain saturated culture, oil extract diluted 1:1000 before measuring the absorption spectrum. 6x 3rd cycle: extraction starting from 5400ml of C. reinhardtii EN1 strain saturated culture in three extraction steps recycling in each step the oil used for extraction to increase ketocarotenoid concentration, oil extract diluted 1:1000 before measuring the absorption spectrum. 6x 4th cycle: extraction starting from 7200ml of C. reinhardtii EN1 strain saturated culture in three extraction steps recycling in each step the oil used for extraction to increase ketocarotenoid concentration, oil extract diluted 1:1000 before measuring the absorption spectrum. 6x 5th cycle: extraction starting from 9000ml of C. reinhardtii EN1 strain saturated culture in three extraction steps recycling in each step the oil used for extraction to increase ketocarotenoid concentration, oil extract diluted 1:1000 before measuring the absorption spectrum. 6x 6th cycle: extraction starting from 10800ml of C. reinhardtii EN1 strain saturated culture in three extraction steps recycling in each step the oil used for extraction to increase ketocarotenoid concentration, oil extract diluted 1:1000 before measuring the absorption spectrum. Oil extraction report on large scale: Oil extraction was performed according to the procedure developed for lab scale starting from 200 gr of total dry biomass (corresponding to a volume of 250L of a saturated cell culture). The overall biomass was extracted in three independent extractions (Extraction 1, 2, and 3), recycling the oil extract for up to 4th cycle, keeping the same ratio of oil/biomass adopted in the lab scale experiment. In the case of Extraction 1 the extraction was performed on wet biomass, while Extraction 2 and 3 were performed on dry biomass. After Extractions 1 and 2, the final pellet obtained was further extracted in oil (samples “ 2nd extraction 1” and “2nd extraction 2”). The spectra recorded are in the sheet “Raw”. In the sheet “Quantification” there are the results of the ketocarotenoids quantification considering the dilution (in oil) applied to the oil extracts. In the sheet “extraction eff." the finalresults in terms of starting material, final oil volume, ketocarotenoid concentration and extraction efficiency are presented. The overall extraction efficiency obtained considering all the extraction performed and the total oil extracts obtained is also reported