Protein separation and characterization from Neochloris Oleoabundans

The process design was made referring all the calculations to a volumetric flow rate of 1000 l/h with a biomass concentration coming out from the photobioreactor of 2g/l. Two different process designs are shown: the first one is meant to the recovery of the only RuBisCO from the water soluble fraction of the proteins while the second is meant to recovery also the proteins stuck into the cell wall debris.

For the first process, harvesting of the biomass is the first required step to increase concentrations and reduce the volume of downstream units. The technology chosen for the harvesting is Tangential Flow Filtration (TFF) with values measured from (Petrusevski, Bolier, Van Breemen, & Alaerts, 1994) for 6 different microalgal strains. Neochloris Oleoabundans was not among the six examined strains, so reference values, reported in Fig.4.2, were assumed for this calculation. With a chlorophyll-α content higher than 2.5μg/l. Chlorophyll-α values were taken from (Pruvost, Van Vooren, Cogne, & Legrand, 2009).

The Tangential Flow Filtration operates with a Concentration Factor “C” equal to 40 and the related biomass loss factor is between 11% and 30% (for our calculation 30% has been assumed). Membrane pores are 0.45μm since smaller pores would cause a more frequent clogging. Two TFF units are required to recover microalgae and recycle water with a lower concentration of biomass . From the first unit the concentrate stream has a total volumetric flow rate of 25 l/h, a mass flow rate of 1.40kg/h and a biomass concentration of 56 g/l. The rest of the inlet flow passes through the membrane and has a flow rate of 975 l/h, mass flow rate of 0.6 kg/h and a concentration of 0.60 g/l.

This stream is fed to a second Filtration unit, where the concentrate has a volumetric flow rate of 24.40 l/h, a mass flow rate of 0.42 kg/h and a biomass concentration of 17.2 g/l The stream passed through the membrane has a flow rate of 950.6 l/h, a mass flow rate of 0.18 kg/h and a concentration of 0.19 g/l and is recycled back to the photobioreactor. The two concentrated streams are mixed together forming a stream with 49.40 l/h, 1.82 kg/h, 36.85 g/l which is then centrifuged at low speed to increase further the biomass concentration and to reduce volume and solvent flow rates of the chromatography separation section. This centrifugation step will be done with a G-force between 10.000 and 13.000g, reaching 22% of solid concentration in the precipitate( according to Christenson & Sims,( 2011). The precipitate is now concentrated up to 220 g/l, with a total volumetric flow rate of 10.12 l/h, composed by 1.82 kg/h of biomass and 8.27 l/h of water. Supernatant from the centrifuge is recirculated to the photobioreactor since it still contains nutrients. Precipitate from the centrifuge is sent to the cell miller in order to break the cell membrane.

One more centrifugation step is required to precipitate cell wall debris and clear the supernatant that contains the water soluble fractions of the proteins. This centrifugation phase should be carried at least at 25.000g, to reach 60% solids (FSA Environmental, 2002). Cell wall debris flow rate is 1.67 kg biomass/h is sent to the fermenter. According to (Collet, Hélias, Lardon, Ras, A., & Steyer, 2011) the biomass can produce 438 l/h of biogas with a heating value of 0.4KW. Values for conversion of the biomass to biogas are valid for Chlorella vulgaris but they are considered to be consistent reference values for Neochloris Oleoabundans since they make part of the same microalgal division (Chlorophyta). Supernatant from the centrifuge is sent to a microfiltration unit with a membrane pore size of 0.2μm to remove completely traces of the cell wall and avoid clogging of the chromatographer. Liquid from the microfiltration is then sent to a dialysis unit where the liquid flows into a membrane system where on the opposite side the equilibration buffer for the HPLC is run.

This operation is required to reduce conductivity of the protein solution, to operate the liquid chromatography in steady starting conditions and to increase the efficiency of the chromatofocusing. The buffer used for salinity decrease is regenerated in a close system with ionic exchange resins. According to (Krokhin & Ying, 2006) dialysis time should be about 6 hours but this value strongly depends on the ions concentration of the cultivating medium. Anionic exchange chromatography has been chosen as the way for protein fractionation. A flow rate of 7.16 l/h of 21.1 g/l proteins has to be fractionated. The target protein for the separation is RuBisCO, that represents 19.96% of the water soluble proteins. The solvent to sample ratio used in lab was 1562 to 1, that is absolutely unbearable on a larger scale. Elution time can be 10 times reduced since isolation of only RuBisCO is required and the solvent required for protein purification decrease to 156.2 times the sample volume meaning a consumption of 1117 l of solvents/h. RuBisCO’s fraction has to be separated from the rest of the proteins eluted. It has to be desalted and then freeze dried to be concentrated to powder. The expected quantity of purified RuBisCO is 30.15 g/h. The scheme explained above represents the process made in laboratory with some adaptations due to the larger scale.