Use of physical methods in the optimization of lipid extraction of Haematococccus pluvialis produced in myxotrophic cultivation aiming to obtain proteins and astaxanthin from residual biomass

The microalgae Haematococcus pluvialis has aroused great interest in its cellular content, mainly astaxanthin and lipids, and is commonly cultivated for the production of biofuel. However, for this production to be sustainable, it is necessary to use efficient lipid extraction methods and the use of residual biomass. The present study aims to evaluate pretreatment methods for the extraction of lipids and to analyze the residual biomass of the microalgae H. pluvialis. The microalgae was cultivated in a mixotrophic and semi-continuous system, being inoculated to 10% of the volume, starting from 40 mL test tubes to 20L volumes, and its growth was monitored through an optical microscope, collecting the biomass in the macrozooide phase (vegetative) and aplanospore (cystic) phase. These samples were centrifuged (3500 g for 15 minutes) and lyophilized, being determined the productivity in dry biomass and submitted to lipid extraction. The extraction was by chemical method, using chloroform:methanol as solvent, and had pre-treatment methods: biomass with chloroform:methanol under ultrasound (BCMU), biomass with chloroform:methanol under γ-radiation (BCMR) and dry biomass under γ-radiation (BSR); in the vegetative and cystic phases of the life cycle, in triplicate. After lipid extraction, the residual biomass was dried and submitted to quantitative analysis of proteins, in the vegetative and cystic phases, and astaxanthin, only in the cystic phase, all in triplicate. The dry biomass yield was 0.135 ± 0.07 g L^-1 in the vegetative phase and 0.317 ± 0.04 g L^-1 in the cystic. The total lipid content was higher in the vegetative phase (P <0.05), with a mean of 0.113 ± 0.02 g and in the cystic phase 0.076 ± 0.02 g, with a mean yield of 16% and 11% of the dry weight, respectively, with the BCMR method being the one with the highest yield in both phases. Regarding the astaxanthin concentration in the residual biomass of the cystic phase, it was observed its preservation using the BSR method (76.2 ± 11.5 mg L^-1), similar to the crude biomass (79.0 ± 6.8 mg L^-1), while the residual biomass of the other methods presented low astaxanthin concentration (BCMU: 10.07 ± 2.0 and BCMR: 10.3 ± 2.3). The protein content was, on average, 44.26 ± 2.29% of the dry weight for the vegetative phase and 19.9 ± 2.17% for the cystic, not significantly different (P> 0.05) for the residual biomass, from the lipid extraction with the different pretreatment methods, and the crude biomass. Therefore, the lipid extraction method with gamma irradiation was efficient for the vegetative cells of H. pluvialis, mainly in biomass with chloroform:methanol (BCMR). However, considering the preservation of astaxanthin in the residual biomass, gamma irradiation in dry biomass (BSR) was the best method. In addition, it was possible to attest that independent of the lipid extraction method, the residual biomass presented as an excellent protein source, in the two phases of the life cycle. This is the first study to use gamma radiation as an algal biomass pretreatment method for lipid extraction. (author)